We want to thank Wladislaw Maier (BMG Labtech GmbH) for his support in establishing the self-written script for the plate reader. This work was supported by the Austrian &#34;Fonds zur F&#246;rderung der wissenschaftlichen Forschung&#34; (FWF) to PM (P24189) and the PhD program &#34;DK Molecular Enzymology&#34; (W901) to PM.

1. Design, Preparation, and Expression of the lux Operon in Escherichia coli
Note: See Table of Materials for information on commercial kits used in this section.
<ol>
	<li>For transferring the lux operon into E. coli choose a standard pET vector with appropriate restriction sites and antibiotic resistance gene of interest (e.g., pET28a; NcoI, XhoI, kanamycin).</li>
	<li>Design the fragments and overlapping primers for Gibson assembly based on the DNA sequence of Photobacterium mandapamensis 27561 (GenBank: DQ988878.2).</li>
	<li>Set up a standard PCR reaction with the designed primers and the isolated genomic DNA of Photobacterium mandapamensis 27561 as template (see Supplemental Material for primers and conditions). 
	NOTE: Isolation of genomic DNA of the respective bacterial strain enhances PCR efficacy.</li>
	<li>Purify the PCR product via spin-column purification.</li>
	<li>Perform a restriction digestion of the isolated pET28a vector with NcoI and XhoI at 37 &#176;C for 45 min.</li>
	<li>Purify the linearized vector and the PCR fragments via agarose gel electrophoresis and subsequent spin-column purification.</li>
	<li>Determine the DNA concentration of each fragment and the linearized vector and calculate the optimal quantities for the assembly according to the protocol20,21. 
	NOTE: Efficacy of assembly depends on fragment size and number and has to be adjusted according to manufacturer&#39;s protocol20,21.</li>
	<li>After combining all fragments and the buffer in a PCR tube, incubate the assembly mixture in a PCR machine at 50 &#176;C for 1 h.</li>
	<li>Transform the assembled vector product according to standard transformation protocols for E. coli bacterial plasmid transformation into an appropriate E. coli system for high yield plasmid replication (e.g., E. coli TOP10 or XL-1).</li>
	<li>Pick colonies from the transformation plate and streak on new plates for DNA isolation.</li>
	<li>Isolate plasmid DNA according to standard protocols.</li>
	<li>To verify the correct assembly of the plasmid including all fragments, first perform a colony PCR according to standard protocols using primers specific for every assembled fragment.</li>
	<li>Additionally to the colony PCR and subsequent agarose gel electrophoresis, prepare all isolated assembly vectors for DNA sequencing to verify the correct assembly and the correct DNA sequences.</li>
	<li>Transform the verified plasmid according to standard transformation protocols for E. coli bacterial plasmid transformation into an appropriate E. coli system for high yield protein production (e.g.,E. coli BL21). 
	NOTE: Continue directly with expression protocol below. For longer storage, the preparation of a glycerol stock is recommended.</li>
</ol>
2. Expression of Modified E. coli Strains
<ol>
	<li>Prepare an overnight culture (ONC) for expression by inoculation of an appropriate volume of LB medium (e.g., 100 mL) with the previously prepared glycerol stock of the E. coli BL21 cells transformed with the assembled plasmid or directly from a transformation plate. Add 100 &#181;L of kanamycin (50 mg/mL; antibiotic resistance gene of pET28a) and incubate the ONC at 37 &#176;C and 120 rpm in an incubator shaker overnight.</li>
	<li>Inoculate the main expression culture (e.g., 800 mL of LB medium) with 8 mL of the ONC and add 800 &#181;L of kanamycin (50 mg/mL).</li>
	<li>Incubate the main culture at 37 &#176;C and 120 rpm in an incubator shaker until the cell density reaches an OD600 of 0.6 - 0.8 (approximately 2.5 h).</li>
	<li>Reduce the incubation temperature to 28 &#176;C.</li>
	<li>Induce protein expression by adding IPTG to a final concentration of 0.1 mM. 
	NOTE: Empirical tests showed that the reduction of the temperature to 28 &#176;C gave the highest light intensity.</li>
	<li>Observe cells until they start shining (approximately 1 h). 
	NOTE: Depending on the purpose of the expression, the cells are grown until the next day and are then harvested, or the cells can be kept shaking as long as they are shining (maximum 48 h). Harvesting the cells and purification of any proteins can be done according to standard procedures.</li>
</ol>
3. Expression of Bacterial Bioluminescent Strains
NOTE: Bacterial bioluminescent strains require specific growth medium/artificial sea water medium for growth and light production.
<ol>
	<li>Prepare artificial sea water medium, composed of two separately prepared medium components. 
	&#8203;NOTE: The preparation of the artificial sea water medium was adapted from the original protocol22. The following amounts are for 1 L liquid medium or 1 L agar medium.
	<ol>
		<li>For artificial sea water medium, weigh in the following salts: 28.13 g NaCl, 0.77 g KCl, 1.60 g CaCl2 &#183; 2H2O, 4.80 g MgCl2 &#183; 6H2O, 0.11 g NaHCO3, and 3.50 g MgSO4 &#183; 7H2O.</li>
		<li>Add 1 L of distilled water and dissolve all components.</li>
		<li>For LB medium, weigh in the following ingredients: 10 g yeast extract, 10 g peptone, and for agar plates, an additional&#160;20 g agar.</li>
		<li>Add 250 mL of tap water and dissolve components.</li>
		<li>Autoclave both prepared media separately at 121 &#176;C for 20 min.</li>
		<li>For agar plates, combine 250 mL of LB medium with 750 mL of artificial sea water medium directly after autoclaving and prepare plates.</li>
		<li>For liquid medium, combine 250 mL of LB medium with 750 mL of artificial sea water medium either directly after autoclaving or when cooled down. 
		NOTE: The artificial sea water medium may get turbid through&#160;salt precipitation.</li>
	</ol>
	</li>
	<li>Streak the bacterial bioluminescent strains on artificial sea water medium agar plates and incubate overnight at 24 - 30 &#176;C. 
	NOTE: Long time storage of bacterial strains is normally achieved through freezing glycerol stocks of the bacterial culture. Strains should always be streaked on agar plates first to assure uniform&#160;starting conditions for all strains, prior to usage for liquid cultures, due to a lag phase in growth after thawing.</li>
	<li>Prepare an ONC by inoculating 100 mL artificial sea water medium with a single colony from the plate. Incubate the ONC at 24 - 30 &#176;C and 120 rpm in an incubator shaker overnight.</li>
	<li>Inoculate 800 mL of artificial sea water medium with 8 mL of ONC.</li>
	<li>Incubate the bacterial cells at 24 - 30 &#176;C and 120 rpm in an incubator shaker. 
	NOTE: The light intensity profile of bioluminescent bacteria strongly varies with temperature. Depending on the regulatory mechanisms of the light production of the respective bacterial strain, light emission may start after approximately 1 - 6 h.</li>
	<li>Observe bacterial cell culture&#160;until they start shining (approximately 1 - 6 h). 
	NOTE: Depending on the purpose of the expression, the cells are grown until the next day and are then harvested, or the cells can be kept shaking as long as they are shining. Harvesting the cells and purification of any proteins can be done according to standard procedures.</li>
</ol>
4. In Vivo Activity Assay for Bacterial Bioluminescent Strains and Modified E. coli Strains
NOTE: Long time storage of strains is normally acieved&#160;through freezing glycerol stocks of the bacterial culture. Strains should always be streaked on agar plates first to assure uniform&#160;starting conditions for all strains, prior to usage for liquid cultures, due to a lag phase in growth after thawing.
<ol>
	<li>Streak the desired bioluminescent bacterial strain or modified E. coli strain on an agar plate and incubate at 28 &#176;C overnight. 
	NOTE: The incubation temperature can vary from strain to strain and has to be evaluated empirically. To be able to compare bioluminescent bacterial strains and modified E. coli strains, growth conditions have to be identical.</li>
	<li>Inoculate 3 mL of medium with the respective strain with a single colony from an agar plate and incubate the cells at 28 &#176;C and 180 rpm in an incubator shaker for approximately 1 - 2 h.</li>
	<li>Measure the cell density of a 1:10 dilution of the liquid culture at 650 nm. Calculate the ratio and volume for 1 mL culture with an OD650 of 0.05. 
	NOTE: The subsequent plate reader assay will determine the cell density at 650 nm to avoid interference by the light emission of the strains.</li>
	<li>Pipette the calculated volume of culture and medium into a 24-well black-walled plate with glass bottom. For the modified E. coli strain, add 1 &#181;L of kanamycin (antibiotic resistance of pET28a vector) and 1 &#181;L of IPTG (induction of gene expression) to the samples. Place a lid on the plate to avoid evaporation during the measurements. 
	NOTE: To assure that the pET28a vector containing the whole lux operon does not get lost by the E. coli culture, kanamycin must be added to each E. coli sample in the plate wells and to assure that light production of the E. coli cells can be measured, the gene expression must be induced by IPTG. To avoid crosstalk and measurement interference, black-walled well plates with glass bottom and transparent lid showed best results. Nevertheless, crosstalk can be observed and well positions have to be chosen carefully.</li>
	<li>Start the measurement in a plate reader. 
	NOTE: The plate reader protocol is based on a script specially developed for this assay (see Supplemental Material) that combines two measurements, absorbance and bioluminescence. Data points are collected every 10 min with permanent shaking between the measurements and a constant temperature of 28 &#176;C.</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+tetradecanal+as+the+natural+aldehyde+in+bacterial+bioluminescence.">Evidence for tetradecanal as the natural aldehyde in bacterial bioluminescence.</a> Proc Natl Acad Sci USA. 76 (1), 265-267 (1979).</li><li>Kurf&#252;rst, M., Ghisla, S., Hastings, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+postulated+structure+of+the+primary+emitter+in+the+bacterial+luciferase+reaction.">Characterization and postulated structure of the primary emitter in the bacterial luciferase reaction.</a> Proc Natl Acad Sci USA. 81, 2990-2994 (1984).</li><li>Dunlap, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemistry+and+genetics+of+bacterial+bioluminescence.">Biochemistry and genetics of bacterial bioluminescence.</a> Biolumin Fundam Appl Biotechnol. 1, 37-64 (2014).</li><li>Meighen, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Bioluminescence:+Organization,+regulation,+and+application+of+the+lux+genes.">Bacterial Bioluminescence: Organization, regulation, and application of the lux genes.</a> FASEB J. 7, 1016-1022 (1993).</li><li>Moore, S. A., James, M. N. G., O'Kane, D. J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+of+Photobacterium+leiognathi+non-fluorescent+flavoprotein+with+limited+sequence+identity+to+bacterial+luciferase.">Crystallization of Photobacterium leiognathi non-fluorescent flavoprotein with limited sequence identity to bacterial luciferase.</a> J Mol Biol. 224, 523-526 (1992).</li><li>Moore, S. A., James, M. N. G., O'Kane, D. J., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+flavoprotein+related+to+the+subunits+of+bacterial+luciferase.">Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.</a> EMBO J. 12 (5), 1767-1774 (1993).</li><li>Moore, S. A., James, M. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+structural+features+of+the+luxF+protein+and+the+subunits+of+bacterial+luciferase:+Evidence+for+a+(&#946;&#945;)8+fold+in+luciferase.">Common structural features of the luxF protein and the subunits of bacterial luciferase: Evidence for a (&#946;&#945;)8 fold in luciferase.</a> Protein Sci. 3, 1914-1926 (1994).</li><li>Moore, S. A., James, M. N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+refinement+of+the+non-fluorescent+flavoprotein+from+Photobacterium+leiognathi+at+1.60+&#197;+resolution.">Structural refinement of the non-fluorescent flavoprotein from Photobacterium leiognathi at 1.60 &#197; resolution.</a> J Mol Biol. 249, 195-214 (1995).</li><li>Kita, A., Kasai, S., Miyata, M., Miki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+flavoprotein+FP390+from+a+luminescent+bacterium+Photobacterium+phosphoreum+refined+at+2.7+&#197;+resolution.">Structure of flavoprotein FP390 from a luminescent bacterium Photobacterium phosphoreum refined at 2.7 &#197; resolution.</a> Acta Crystallogr Sect D Biol Crystallogr. 52 (1), 77-86 (1996).</li><li>Bergner, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+biochemical+properties+of+LuxF+from+Photobacterium+leiognathi.">Structural and biochemical properties of LuxF from Photobacterium leiognathi.</a> Biochim Biophys Acta - Proteins Proteomics. 1854 (10), 1466-1475 (2015).</li><li>Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W., Bossier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing+and+quorum+quenching+in+Vibrio+harveyi:+Lessons+learned+from+in+vivo+work.">Quorum sensing and quorum quenching in Vibrio harveyi: Lessons learned from in vivo work.</a> ISME J. 2 (1), 19-26 (2008).</li><li>Meighen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+bacterial+bioluminescence.">Genetics of bacterial bioluminescence.</a> Annu Rev Genet. 28, 117-139 (1994).</li><li>Kelkar, M., De, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+based+in+vivo+screening+technologies.">Bioluminescence based in vivo screening technologies.</a> Curr Opin Pharmacol. 12 (5), 592-600 (2012).</li><li>Waidmann, M. S., Bleichrodt, F. S., Laslo, T., Riedel, C. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+luciferase+reporters:+The+Swiss+army+knife+of+molecular+biology.">Bacterial luciferase reporters: The Swiss army knife of molecular biology.</a> Bioeng Bugs. 2 (1), 8-16 (2011).</li><li>Wilson, T., Hastings, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioluminescence living lights, lights for living. , (2013).</li><li>Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., Smith, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Gibson, D. G., Smith, H. O., Hutchison, C. A., Venter, J. C., Merryman, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+synthesis+of+the+mouse+mitochondrial+genome.">Chemical synthesis of the mouse mitochondrial genome.</a> Nat Methods. 7 (11), 901-903 (2010).</li><li>Gibson, D. 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The authors have nothing to disclose.

The construction of an expression plasmid containing four fragments composing the whole lux-rib operon of P. mandapamensis 27561 was achieved via Gibson cloning. This E. coli based lux gene expression enables E. coli cells to emit light. Avoiding quorum sensing regulation and non-standard growth conditions are substantial advantages of this system.
The advantages of using Gibson cloning strategy are the easy assembly of multiple linear DNA fragments, high flexibility and no need for specific restriction sites18,19,20. This method allows to easily change a lux operon, excluding single genes or gene clusters or introducing new genes or exchanging genes from one strain with another. A prerequisite for the application of this method is the availability of the respective lux operon gene sequence. Only for a small number of bioluminescent bacteria is the complete DNA sequence of the respective lux operon known and/or available. Many of these sequences are fragmented because they were generated using shotgun sequencing. In the case of the investigated P. mandapamensis 27561 the complete DNA sequence of the lux operon is known and available from the NCBI gene database (GenBank: DQ988878.2).
One critical step in the protocol of the Gibson assembly is the calculation of the DNA concentration of the single fragments. In the assembly protocol of the manufacturer it was recommended to use 0.2 - 0.5 pmols and a total volume of 20 &#181;L for 4 - 6 fragments20,21. In our case, where luxCDABFEG-ribEBH is comprised of 4 fragments, the concentrations and total volumes were 0.1 pmol and 45 &#181;L, respectively, depending on the yield of PCR products. On the one hand, the concentration had to be reduced and on the other hand, the volume had to be increased. Nevertheless, the assembly worked very well and DNA sequencing confirmed the correct insertion at the first attempt. This finding confirmed Gibson assembly as a robust and appropriate method for our investigations, which can easily be modified18,19,20.
The newly established plate reader assay is an easy to handle method. It enables a simple primary analysis of new or (un-)known bioluminescent bacterial strains and gives already a first hint on the regulatory mechanism of light production (e.g., lag in luminescence at low cell densities). Additionally, growth conditions in combination with light production can easily be evaluated by simply changing growth medium or temperature.
The measurements with the plate reader were performed at 28 &#176;C. The reason for this unusual setting is the temperature sensitivity of bioluminescent bacterial strains, where temperatures above 30 &#176;C lead to less or even no growth and/or light emission. Note, that the plate reader is capable of keeping a defined temperature over time with the limitation of a missing active cooling system. Therefore, the ambient temperature has to be below the temperature of the measurement.
As a proof of concept, the comparison of the E. coli construct with the bioluminescent strain P. mandapamensis (Figure 5) on the one hand and the reference measurements (Figure 4) on the other hand were performed and confirm the reliability of this newly established system. Additionally, the long-term measurement assured the longevity of the light emission (Figure 6). But one has to consider that OD values are not reliable above a certain optical density, depending on the characteristics of the used measurement device (approximately 15 h in the present case) where an appropriate dilution would be needed for a measurement in the linear range of the detector. These high variances in OD values make these long time measurements not reliable. Therefore, shorter measurements such as 10 h are recommended using the experimental setup reported here.
Limitations of the established plate reader assay can be seen in Figure 7. The difficulty is to find an appropriate setting of the measurement. The &#39;gain value&#39; adjusts the sensitivity of the photo multiplier tube (PMT). The gain is the amplification of the signal in the PMT, meaning that a higher gain factor will increase the signal. If the gain is set too low, the signal to noise ratio becomes bigger and light intensities of &#34;low&#34; shining bioluminescent bacteria cannot be measured any more (signals close to zero, data not shown). The challenge in a bioluminescent assay is to set the gain so the measurement results for all bacterial strains stay within the range of the instrument. Additionally, the measurement range for luminescence depends on the measurement interval time (e.g., maximum of 2,000,000 for 1 s).
The gain was set to 2800, which was empirically tested and chosen for the specific plate reader used for the establishment of this method. The used gain setting allows the recording of maximum emitted light by the bioluminescent E. coli system, P. mandapamensis 27561, and P. mandapamensis S1 without an overflow, but for the strains P. mandapamensis TH1 and V. harveyi 14126 the gain was too high. Therefore, these latter strains exceed the detection limit and the real maximal light intensity cannot be measured. This technical limitation might prevent the comparison of bioluminescent bacteria, which show high variations in maximum light emission, although growth conditions and cell densities might be comparable.
Positioning of the analyzed bacterial strains within the used well plates has to be evaluated empirically. Although black well plates with glass bottoms were used, crosstalk between the samples was observed. The light intensity of specific strains is so high, that all neighboring wells will show false positive light emissions (e.g., blank). Therefore, it is important to measure two different strains either separately or with a certain spatial separation to each other.
There are already many modified E. coli strains known that contain parts of the lux operon and are mainly application-oriented15,16,17. The methods described here, aim at fundamental research, for example the possibility of analyzing each lux gene separately. Although research of bioluminescence has a long history, there are still many open questions. By excluding or introducing genes from the lux operon, exchanging the luxAB genes with genes from another strain, or analyzing protein complexes, embedded in the easy to handle E. coli system and further application of the plate reader assay, it might be possible to gain more information on regulatory processes and the functions of lux genes.

The emission of light by living organisms (bioluminescence) is a fascinating process found in bacteria, fungi, insects, nematodes, fish, and squids1. Bioluminescent light emission emerges from a chemiluminescent reaction in which chemical energy is (partially) transformed into light energy ("cold light"). In bioluminescent bacteria, the heterodimeric enzyme luciferase catalyzes the monooxygenation of long chain, aliphatic aldehydes, such as tetradecanal, to the corresponding acids accompanied by light emission with a maximum at 490 nm2,3.
<img alt="Bioluminescence reaction diagram; luciferase enzyme converts substrate, emitting light (~490 nm)." class="xfigimg" src="/files/ftp_upload/57881/57881fig1v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: General reaction scheme of bacterial luciferase. The bacterial luciferase (LuxAB) catalyzes the monooxygenation of long chain aldehydes (CH3(CH2)nCHO) by utilizing reduced flavin mononucleotide (FMNH2) and molecular oxygen (O2), yielding the products long chain acids (CH3(CH2)nCOOH), flavin mononucleotide (FMN), water (H2O), and the emission of light centered at 490 nm. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The energy released during this oxidation causes an excited state FMN-4a-hydroxide, which serves as the light emitting luciferin4. The proteins involved in bacterial bioluminescence, namely LuxCDABEG, are encoded by the lux operon and are highly conserved over various bacterial strains2,5. The genes luxA and luxB encode for the heterodimeric luciferase; luxC's, luxD's, and luxE's gene products are components of a fatty acid reductase complex; and luxG encodes for a flavin reductase6. A number of bioluminescent Photobacteria (e.g.,Photobacterium mandapamensis 27561) carry the additional luxF gene. It was reported that LuxF is a homodimeric protein that binds the unusual flavin derivative 6-(3'-(R)-myristyl)-FMN (myrFMN)7,8,9,10,11,12. Additional genes have been identified that are responsible for riboflavin synthesis (e.g., ribEBH) and furthermore regulatory genes have been reported, that play a role in quorum sensing regulation of bioluminescence, especially for Vibrio fischeri and Vibrio harveyi6,13. Despite the highly conserved gene order, bioluminescent bacteria show high variations in characteristics like growth behavior, intensity of light emission, or regulation of bioluminescence2,5,14.
Several modified strains or plasmids containing parts or whole lux operons are known, exploiting bioluminescence as reporter systems. Various applications such as determining promoter activity, monitoring of bacterial contaminations in environment or food samples, Bioluminescence Resonance Energy Transfer (BRET), in vivo imaging of infections in eukaryotic organisms, pyrosequencing, and so forth were established15,16,17. Interestingly, the three most frequently used bioluminescent reporter systems are derived from the North American firefly (Photinus pyralis), the enteric pathogen of nematodes (Photorhabdus luminescens), and the sea pansy (Renilla reniformis). None of those systems has a bacterial origin, but the use of lux genes and operons from bacterial origins is gaining more interest for applied research16. The less abundant application of bioluminescence proteins from bacterial sources is mainly due to lower stability and longevity of bacteria derived luminescent proteins which can be related to their marine habitats. Bioluminescent bacteria of marine habitats are not cultivable under standard lab conditions. These bacteria require specific growth media and conditions, such as artificial sea water medium and lower growth/incubation temperatures (e.g., 28 °C).
To simplify comparison of lux operon characteristics or single lux genes of a range of different bioluminescent bacterial strains, a method to standardize lux operon expression and analysis is a prerequisite. Thus, the idea of integrating the whole lux-rib operon into the standard-research bacterium Escherichia coli (E. coli) emerged. For this purpose, Gibson assembly proved to be a useful tool to integrate multiple linear, overlapping fragments into one expression vector without the need for specific restriction sites. This method is also suitable when DNA inserts are too large (e.g.,P. mandapamensis 27561 luxCDABFEG-ribEBH; ~ 9 kb operon size) to be amplified via PCR. A lux operon can be separated into multiple overlapping fragments, then be assembled into one expression plasmid and finally the sequence verified assembly product can be directly transformed into an appropriate E. coli system for high yield protein expression18,19,20. In addition to the easy to handle E. coli based lux gene expression, a simple method combining recording of cell growth and bioluminescent light emission remained to be established. The method described here allows the in situ measurement and correlation of cell density and light emission of bioluminescent bacteria.
The analysis of lux genes and lux operon order and regulation of various bioluminescent bacteria with, on the one hand, an artificial bioluminescent E. coli system containing the whole lux-rib operon of P. mandapamensis 27561 and, on the other hand, a newly developed plate reader assay combining the in situ recording of cell density and light emission, helps to gain more information on the various bacterial lux systems. This fundamental characterization and comparison of luciferases and related enzymes may lead to alternatives to the already established reporter systems with enhanced stability and activity.

<table><tbody><tr><td>NEBuilder High-Fidelity DNA Assembly Cloning Kit</td><td>New England Biolabs Inc.</td><td>E2621S</td><td>for 10 rxn&lt;br/&gt; dx.doi.org/10.17504/protocols.io.cwaxad</td></tr><tr><td>Phusion Polymerase&amp;nbsp;</td><td>Thermo Scientific</td><td>F530S</td><td></td></tr><tr><td>Q5 High Fidelity DNA Polymerase</td><td>New England Biolabs Inc.</td><td>M0491S</td><td></td></tr><tr><td>GeneJet Genomic DNA Purififcation Kit</td><td>Thermo Scientific</td><td>K0721</td><td>for 50 rxn</td></tr><tr><td>Restriction enzymes and buffer (NcoI, XhoI)</td><td>New England Biolabs Inc.</td><td>R3193S/R0146S</td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up kit&amp;nbsp;</td><td>Promega</td><td>A9282</td><td>for 250 rxn</td></tr><tr><td>Monarch DNA Gel Extraction Kit&amp;nbsp;</td><td>New England Biolabs Inc.</td><td>#T1020S</td><td>for 50 rxn</td></tr><tr><td>GeneJet Plasmid Miniprep Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>K0503</td><td>for 250 rxn</td></tr><tr><td>GoTaq G2 DNA Polymerase</td><td>Promega</td><td>M7841</td><td></td></tr><tr><td>24-well black sensoplate with glass bottom</td><td>Greiner Bio One</td><td>662892</td><td></td></tr><tr><td>FLUOStar Omega plate reader&amp;nbsp;</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>OPTIMA/Mars Analysis Software</td><td>BMG Labtech</td><td></td><td>Version 2.20</td></tr><tr><td>Microsoft Excel 2010</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Multitron Standard Incubator Shaker</td><td>Infors AG</td><td></td><td></td></tr></tbody></table>

in situ measurement and correlation of cell density and light emission of bioluminescent bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

The gene order of the lux operon - luxCDABFEG - is highly conserved over various strains2,5,14. For the design of the plasmid, the sequence information was taken from the bioluminescent bacterial strain Photobacterium mandapamensis 27561 and its gene order was kept the same, and, also, noncoding sequences between single genes were considered. A schematic overview of the applied Gibson cloning strategy is depicted in Figure 2. Four fragments in total, luxCDAB, luxF, luxEG, and ribEBH, with 20 - 40 base pair overlapping sequences were generated. After following all steps of the Gibson assembly20, DNA sequencing confirmed the correct assembly of the plasmid, including all fragments. The vector map of the final assembly product pET28a containing the lux-rib operon is depicted in Figure 3. A significant advantage of this modified pET28a vector is the utilization of standardized E. coli growth conditions and controlled induction with IPTG.
To measure light emission of bioluminescent bacteria and the respective cell density, a plate reader based method was developed. The method for the plate reader was generated combining single measurement scripts for light intensity and cell density. This novel script enabled the measurement of OD650 and light intensity every 10 min for a user defined time frame, which has to be adjusted to the generation time of the bacteria used for the respective analysis (e.g., 10 h). The measurement of the optical density was performed at 650 nm to avoid interference with the light emission. As a proof of concept and to assure the health and the correct growth behavior of the E. coli cells, reference measurements were performed. In Figure 4 the comparison of E. coli BL21 cells, E. coli BL21 cells containing an empty pET28a vector, and E. coli BL21 cells containing the pET28a vector with the lux-rib operon insert are presented. For the latter strain, no IPTG was added to analyze the light emission due to the leakiness of the T7 promoter. All three reference measurements show a sigmoidal growth curve with three growth phases (lag, exponential, and stationary phase). Only the E. coli BL21 cells containing the pET28a vector with the lux-rib operon insert start to emit light, but in contrast to the measurements where expression is induced by addition of IPTG and light is emitted after 30 min, the non-induced cells only start to shine after approximately 5 h and show a much lower light emission (ca. 4-fold) compared to the induced system.
Figure 5 gives a comparison of growth curves and light intensities of the lux operon expressed in E. coli and the bioluminescent bacterial strain P. mandapamensis 27561, either in LB medium or in artificial sea water medium, using the novel established in situ method. To compare these bacteria, the measurements were performed at an incubation temperature of 28 °C. This temperature decreases the growth rate of the modified E. coli strain in LB medium as well as artificial sea water medium, but for bioluminescent bacterial strains lower temperatures are crucial. This temperature dependence is visible in Figure 5A, as P. mandapamensis 27561 shows a much higher cell density than E. coli. Furthermore, while for E. coli strains, LB medium allows generation of higher cell densities, for natural bioluminescent bacterial strains, artificial sea water medium is preferred and essential for bioluminescence. The recorded cell densities correlate with the respective light intensities, as shown in Figure 5B. Noteworthy, the bioluminescent E. coli cells reach similar light emission maxima in both LB medium as well as artificial sea water medium, although the highest intensities were recorded at different time points. In contrast to this observation, P. mandapamensis 27561 is viable with highly reduced growth rates in LB medium, but the bacterial cells did not emit light at all (Figure 5B). In artificial sea water medium, P. mandapamensis 27561 shows a maximum of light emission at around 1 x 104 counts per second, which is nearly a factor of 200 lower than E. coli. Figure 5C represents relative light units where the bioluminescence is normalized by the OD. These results confirm that not only was the insertion of a plasmid containing the lux operon into E. coli successful and functional, but also that this modified E. coli strain is a valid alternative with even higher light emission yields and without the limitation of bacterial bioluminescence of marina bacteria, such as a complex seawater medium and lower temperatures.
Additionally, a long-term measurement of the E. coli based lux-rib gene expression over 24 h was performed to analyze the longevity of the light emission (Figure 6). Light emission lasted for 19.5 h, much longer than the bacterial strains (e.g., P. mandapamensis 27561) where a gradual decrease was observed resulting in very low light emission after 10 h.
To illustrate the limitations of the developed assay, Figure 7 shows the measurement results of three bioluminescent bacterial strains, namely Photobacterium mandapamensis S1, Photobacterium mandapamensis TH1 and Vibrio harveyi 14126. For the first strain (S1), the method works very well and shows a maximal bioluminescence intensity of nearly 2 x 106. For the other two strains (TH1 and 14126), the maximal light intensity cannot be determined because the light intensity generated by both exceeded the detection limit of the used instrument settings. The gain value defined for the developed method (script) for these two strains was set too high. Nevertheless, the onset of bioluminescence activity can be compared with each other. P. mandapamensis TH1 and P. mandapamensis S1 start shining after approximately 1 h and an OD650 value of 0.1 - 0.2, respectively. In contrast, V. harveyi 14126 starts to emit light after approximately 5.5 h at an OD650 value of 1.0. The observed onset of light emission is accompanied by an exponential increase in OD as well as bioluminescence. It is known that bioluminescence of V. harveyi 14126 underlies quorum sensing regulation and therefore a specific cell density allowing the activation of the lux operon, which can be clearly observed in Figure 713. This result demonstrates that with this novel in situ plate reader assay it is possible to easily compare bioluminescent bacteria and also roughly define a regulatory mechanism of these strains by determining whether a quorum sensing regulation can be observed or not.
Figure 8 shows an example of an expression culture of E. coli BL21 cells harboring the assembled pET28a plasmid containing the lux operon after induction with IPTG. After approximately one h after induction, the E. coli cells start shining with a blue-green color. Figure 8A shows the E. coli expression culture photographed in the light and Figure 8B gives the same culture in the dark. Figure 8C depicts an agar plate of artificial sea water medium with P. mandapamensis S1 glowing in the dark in the same blue-green color characteristic for bacterial bioluminescence.
<img alt="Gene cloning process with PCR, restriction digestion, Gibson assembly, plasmid replication, sequencing." class="xfigimg" src="/files/ftp_upload/57881/57881fig2v3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Schematic representation of the applied Gibson cloning strategy. Step (I): Overlapping primers (colored arrows; ca. 20 - 40 base pair overlap) are designed. Overlapping primers contain annealing sequences consisting of the respective 5' and 3' region of one fragment and the respective 3' and 5' region of the adjacent segment. Step (II): The designed fragments for assembly are generated via standard PCR reactions. Step (III): The target vector is linearized by restriction digestion (e.g., NcoI, XhoI). Step (IV): The DNA concentrations of all fragments and the linearized vector have to be determined to adjust concentration appropriate for Gibson assembly (according to the manufacturer's protocol). Step (V): All fragments and the linearized vector with optimized DNA concentrations are combined with the Gibson assembly master mix (T5 exonuclease, DNA polymerase, and DNA ligase) and are incubated at 50 °C for 1 h. Step (VI): The assembly product is transformed according to standard protocols into an appropriate E. coli strain for high yield plasmid replication (e.g., E. coli TOP10 or XL-1). Step (VII): To verify the correct assembly of the plasmid, DNA sequencing of the assembled plasmid has to be performed. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Circular plasmid map, lux operon in pET28a, genes, promoters, 14,625 bp, educational diagram." class="xfigimg" src="/files/ftp_upload/57881/57881fig3v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Vector map of pET28a containing the lux-rib operon. The lux-rib operon of P. mandapamensis 27561 is inserted in the multiple cloning site of pET28a in the original gene order (luxCDABFEG-ribEBH). Restriction sites used for cloning are NcoI and XhoI. Fragments used for Gibson assembly of the operon are luxCDAB in orange, luxF in green, luxEG in blue and ribEBH in lavender; genes within a fragment are shown as a separate box. Noncoding sequences between each gene of the operon are included in the applied cloning strategy. The final plasmid size of pET28a containing the whole lux-rib operon is 14,625 base pairs. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Growth and bioluminescence of BL21 cells with pET28a lux-rib operon; time-course graph." class="xfigimg" src="/files/ftp_upload/57881/57881fig4v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Comparison of growth curves and light intensities of reference strains. The OD at 650 nm and the bioluminescence intensity in counts per second were measured every 10 min over 10 h at 28 °C. All measurements are mean values of three biological replicates with four technical replicates each. Error bars represent standard deviations. E. coli BL21 cells (grey squares), E. coli BL21 cells containing an empty pET28a vector (grey circles), and E. coli BL21 cells containing the pET28a vector with the lux-rib operon insert (black diamond) were analyzed to assure correct growth behavior of our E. coli cells. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lux operon expression analysis: OD, bioluminescence, RLU/OD graphs for E. coli, P. mandapamensis." class="xfigimg" src="/files/ftp_upload/57881/57881fig5v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Comparison of growth curves and light intensities of the lux operon expressed in E. coli (squares) and P. mandapamensis 27561 (circles) in LB medium (open symbols) or artificial sea water medium (filled symbols). All measurements are mean values of three biological replicates with four technical replicates each. Error bars represent standard deviations. All experiments were performed at an incubation temperature of 28 °C. (A) Optical density (OD) measurements at 650 nm were performed every 10 min for 10 h. E. coli lux operon expression (left panel) is compared to P. mandapamensis 27561 (right panel) in LB medium and artificial sea water medium. Cell densities are determined at 650 nm to avoid bioluminescence-interference. (B) Measurement of light intensity (bioluminescence [counts/s]) was performed every 10 min for 10 h. E. coli lux operon expression (left panel) is compared to P. mandapamensis 27561 (right panel) in LB medium and artificial sea water medium. (C) Relative light intensities (RLU/OD) of the lux operon expressed in E. coli (left panel) and P. mandapamensis 27561 (right panel) are determined by normalizing bioluminescence to cell density. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microbial growth chart showing OD and bioluminescence over time; data analysis for bioexperiment." class="xfigimg" src="/files/ftp_upload/57881/57881fig6v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Comparison of growth curves and light intensities of E. coli based lux gene expression for 24 h. The OD at 650 nm and the bioluminescence intensity in counts per second were measured every 10 min over 24 h at 28 °C. All measurements are mean values of three biological replicates with four technical replicates each. Error bars represent standard deviations. Additionally, the relative light intensities (RLU/OD) where bioluminescence is normalized by cell density are represented. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microbial growth and bioluminescence graph; optical density, bioluminescence, and RLU/OD over time." class="xfigimg" src="/files/ftp_upload/57881/57881fig7v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: Comparison of bioluminescent bacteria to evaluate potential quorum sensing regulation. Light emission and cell density are measured every 10 min for 10 h and represent mean values of three biological replicates with four technical replicates each. Error bars represent standard deviations.Measurements of Photobacterium mandapamensis TH1 (black squares), Vibrio harveyi 14126 (grey circles) and Photobacterium mandapamensis S1 (grey diamonds) were compared to each other; (A) depicts the optical density (OD) at 650 nm, (B) the light intensity (bioluminescence [counts/s]), and (C) relative light intensities (RLU/OD). <a href="https://www.jove.com/files/ftp_upload/57881/57881fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bioluminescence process: glowing bacteria culture, fluorescence emission, chemical structure diagram." class="xfigimg" src="/files/ftp_upload/57881/57881fig8.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Bioluminescence in liquid media and on agar plates. (A) 5 L flask with 2 L LB medium inoculated with E. coli BL21 cells expressing the pET28a lux operon plasmid photographed in light. (B) The same culture as in (A) photographed in the dark. Pictures A and B were taken approximately 2 h after induction of expression. (C) Artificial sea water medium agar plate with streaked culture of P. mandapamensis S1 photographed in the dark. <a href="https://www.jove.com/files/ftp_upload/57881/57881fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria at JoVE.com

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

in-situ-measurement-correlation-cell-density-light-emission

Research

JoVE Journal

Biochemistry

11.6K Views.  Graz University of Technology. Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

Video: In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

Genetics

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Immunology and Infection

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

Biology

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

Optimizing Human Intestinal Enteroids for Circadian Rhythm Studies

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

Author Spotlight: In Vitro Investigations of Circadian Rhythms in Multicellular Systems

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening

Source: Hu, Z. et al. <a href="https://www.jove.com/v/62312/a-rapid-screening-workflow-to-identify-potential-combination-therapy">A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.</a>&#160;J. Vis. Exp.&#160;(2021)
In this video, we describe a method highlighting the utility of bioluminescence assay for sensitive and rapid screening of drugs. These drugs can be tested alone or in combination to assess the synergism in their mode of action.

Source: Hu, Z. et al. A Rapid Screening Workflow to Identify Potential Combination Therapy for GBM using Patient-Derived Glioma Stem Cells.&#160;J. Vis. ...

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Summary

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Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Cellular Bioluminescence Based Assay: A High Throughput Method for Combinatorial Drug Screening