We are grateful to Michael Feiss and Jean Sippy for the guidance on phage creation and purification. We thank Michael Elowitz for providing the cell recognition software, Schnitzcell. Work in the Golding lab is supported by grants from the National Institutes of Health (R01GM082837), the National Science Foundation (082265, PFC: Center for the Physics of Living Cells), the Welch Foundation (Grant Q-1759) and Human Frontier Science Program (RGY 70/2008).

<ol>
	<li>Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., Adhya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switches+in+bacteriophage+lambda+development.">Switches in bacteriophage lambda development.</a> Annu. Rev. Genet. 39, 409-429 (2005).</li><li>Ptashne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A genetic switch : phage lambda revisited. , (2004).</li><li>Hendrix, R. 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> Lambda II. , (1983).</li><li>Hershey, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Bacteriophage lambda. , (1971).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decision+making+at+a+subcellular+level+determines+the+outcome+of+bacteriophage+infection.">Decision making at a subcellular level determines the outcome of bacteriophage infection.</a> Cell. 141, 682-691 (2010).</li><li>Edgar, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteriophage+infection+is+targeted+to+cellular+poles.">Bacteriophage infection is targeted to cellular poles.</a> Mol. Microbiol. , (2008).</li><li>Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current protocols in molecular biology. , (1994).</li><li>Sambrook, J., Russell, D. 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> Molecular cloning : a laboratory manual. , (2001).</li><li>Fasman, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical handbook of biochemistry and molecular biology. , (1989).</li><li>Kaiser, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+internal+structure+of+bacteriophage+lambda.">On the internal structure of bacteriophage lambda.</a> J. Gen. Physiol. 49, 171-178 (1966).</li><li>Kobiler, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinetic+analysis+of+the+bacteriophage+lambda+genetic+network.">Quantitative kinetic analysis of the bacteriophage lambda genetic network.</a> Proc Natl Acad Sci. 102, 4470-4475 (2005).</li><li>Shaner, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nat. Biotechnol. 22, 1567-1572 (2004).</li><li>Zeng, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Personal communication with M. Feiss. , .</li><li>Schwartz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adsorption+of+coliphage+lambda+to+its+host:+effect+of+variations+in+the+surface+density+of+receptor+and+in+phage-receptor+affinity.">The adsorption of coliphage lambda to its host: effect of variations in the surface density of receptor and in phage-receptor affinity.</a> J. Mol. Biol. 103, 521-536 (1976).</li></ol>

No conflicts of interest declared.

Bacterial Strains, Phage and Plasmids:
Strain LE392 is supF. It was chosen to suppress the Sam7 mutation in the phage genome (see Table 1 for details). Thus, induced lysogens will eventually lyse and release phage particles, as will infected cells that have chosen the lytic pathway. Lysogenic cells are grown at 30&deg;C due to the presence of the temperature sensitive cI857 allele in the phage genome. After heat induction, gpD-EYFP and wild type gpD are co-expressed from the genome of &lambda;LZ1 and the plasmid pPlate*D respectively. As a result, the capsid of the newly created phage &lambda;LZ2 contains a mixture of gpD-EYFP and gpD proteins. This mosaic phage is structurally stable and sufficiently fluorescent to allow detection of individual phages5. pPRE-mCherry is a reporter plasmid used to detect choice of the lysogenic pathway. The promoter PRE is activated by CII during the establishment of lysogeny1,11. pPRE-mCherry5 was derived from pE-gfp11 by replacing gfp with mCherry12. For more details see our earlier work5.
Growth Condition Parameters:
During lysogen induction (Section 1), mild shaking at 180 rpm gives a good virus yield13. Use of glucose in the growth medium should be avoided as glucose metabolism generates acidic metabolic products, and mature lambda particles are unstable at acidic pH13. The addition of MgSO4 is aimed at stabilizing the phage capsid3. For phages carrying wild type cI (instead of cI857), the lysogen can be induced using the DNA-damaging agent Mitomycin C3. In Step 1.3, the incubation at 37&deg;C should normally not exceed 90 minutes. It is useful to check the cell density by OD600 every 30 min. For a good lysate, OD600 drops to around 0.2 or less, and the remaining OD600 is a result of cell debris. Incubating too long may result in a lower phage yield since the newly created phage may start to inject their DNA into cell debris. To obtain a visible phage band (at least 1 x 1011 phage particles) in Steps 2.11 and 2.13, grow at least 500 ml culture in Step 1.2. The addition of 0.2% Maltose into the growth medium in Steps 5.1 and 5.2 is aimed at inducing expression of LamB, the receptor for phage lambda adsorption3,14. The 1000-fold dilution instead of 100-fold in Step 5.2 is aimed at reducing the mCherry background level from the reporter plasmid pPRE-mCherry. In Step 5.5 for phage DNA injection triggering, 35&deg;C is chosen to avoid induction of the temperature-sensitive cI857 allele.
Phage Purification:
The phage purification steps (Steps 2.1 through 2.11) can be replaced with other purification protocols5, but the final ultracentrifugation through CsCl equilibrium gradient (Steps 2.12 and 2.13) is unavoidable. Swinging bucket rotors are needed in Steps 2.10 and 2.12 to ensure sharp visible phage bands. Obtaining a pure phage stock can easily take up to a week, so it is necessary to check the phage titer along the way to make sure nothing goes wrong during the intermediate steps.
Phage Handling:
During all purification procedures in Section 2, it is critical to handle phage lysate gently to avoid shearing phage tails from phage heads. During cell infection in Section 5 (e.g., Steps 5.5 through 5.7), it is also critical to avoid the shearing of phage particles from the infected cell. Note that if the phage is sheared from the infected cell after injecting its DNA, the result is a "dark" infection, i.e. the infection outcome will be observed in experiment but the infecting phage will not. To minimize such problems, we use a wide pipette tip whenever handling phages or the phage/cell mixture.
DAPI Testing:
Staining the phage stock with DAPI (Section 4) is a quick and efficient method to check the purity of the phage stock. It can also be used to test for possible degradation of an existing phage stock over time. For a pure stock, the co-localization of YFP and DAPI signals under the fluorescence microscope should be close to 100%. We typically observe that less than 1% of the YFP spots do not contain DAPI (representing capsids without the viral genome), which indicates that these particles did not successfully package the viral DNA or had already injected their DNA elsewhere. Less than 1% of the DAPI spots do not contain YFP (corresponding to non-fluorescent phages). If this is not the case, Steps 2.12 through 2.14 need to be repeated in order to purify again. With regards to imaging parameters, the microscope setup in Step 4.3 is not as critical as in Section 5 because no long-term live-cell imaging is required here. However, keeping the same microscopy settings as in Section 5 is useful if one wishes to calibrate the fluorescence intensity of a single phage particle. If the PBS-agarose slab is not very clean, or too much DAPI dye is used, some DAPI spots corresponding to phage DNA may be surrounded with a "halo". If too little DAPI dye is used, the signal from the DAPI channel may be very weak.
Microscope System:
For the imaging in Section 6, we use a commercial inverted epifluorescence microscope (Eclipse TE2000-E, Nikon) with a 100x objective (Plan Fluo, numerical aperture 1.40, oil immersion) and standard filter sets (Nikon). The fluorescence light source is an Arc lamp with control of light intensity. The following features are computer controlled: x, y and z position; bright field and fluorescence shutters; and fluorescence filter choice. An auto-focus feature is required. Otherwise, the focus may easily drift away during the time-lapse movie (normally 4 hours long). The ability to acquire multiple (x,y) positions at each time point is useful, as it allows to follow multiple infection events in parallel. We typically acquire 8 stage positions in each movie, following up to 100 infection events. The camera we use is a cooled 512x512 CCD with 16x16 &mu;m pixel camera with a dynamic range of 16 bits (Cascade512, Photometrics). Acquisition is performed using MetaMorph software (Molecular Devices). The microscope should be placed in a temperature-controlled room; alternatively, the microscope stage should be surrounded by a temperature-controlled chamber.
Image Acquisition: 
For live-cell imaging, it is critical to avoid unnecessary exposure of the sample, which could lead to bleaching and phototoxicity. Therefore, it is best to first characterize your system to find an optimal light exposure which allows for fluorescence detection while not leading to excessive bleaching or inhibiting cell growth. To obtain a good fluorescence image, play with the exciting light intensity, exposure time and camera gain. In Steps 6.2-6.3, the 10 min frame interval is chosen for the purpose of minimizing light exposure. In each frame, only a single in-focus image is needed in phase-contrast (for cell recognition) and fluorescent channels (for determining cell fate). In the first time point, however, multiple z-position images through the YFP channel are required to capture all infecting phages on the cell surface. The YFP exposure time in the initial frame may also need to be higher than that used for the time-lapse movie in the later time frames.
Image Analysis:
Very carefully count phage particles around the cell surface in Step 7.1. As noted above, we take a series of z-stacks through YFP channel in Step 6.2. However, this may still leave some fluorescent phage particles out-of-focus, which challenges the counting. The cell length in the initial time frame is measured using the Metamorph software. The cell length can also be measured by ImageJ or other software tools. Additionally, an automated home built Matlab program can be very useful in obtaining information such as fluorescence change over time along cell lineages.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Fisher Scientific</td>
 <td>C298-500</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Fisher Scientific</td>
 <td>S271-3</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Sigma</td>
 <td>D4527-10KU</td>
 </tr>
 <tr>
 <td>RNase</td>
 <td>Sigma</td>
 <td>R4642-10MG</td>
 </tr>
 <tr>
 <td>PEG8000</td>
 <td>Fisher Scientific</td>
 <td>BP233-1</td>
 </tr>
 <tr>
 <td>SM buffer</td>
 <td>Teknova</td>
 <td>S0249</td>
 </tr>
 <tr>
 <td>NZYM</td>
 <td>Teknova</td>
 <td>N2062</td>
 </tr>
 <tr>
 <td>CsCl</td>
 <td>Sigma</td>
 <td>C3011-250G</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Becton Dickinson</td>
 <td>309585</td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>Becton Dickinson</td>
 <td>305176</td>
 </tr>
 <tr>
 <td>Dialysis cassette</td>
 <td>Thermo Scientific</td>
 <td>66333</td>
 </tr>
 <tr>
 <td>Microscope slide</td>
 <td>Corning</td>
 <td>2947-75x50</td>
 </tr>
 <tr>
 <td>Agarose</td>
 <td>Fisher Scientific</td>
 <td>BP160-100</td>
 </tr>
 <tr>
 <td>SW40Ti ultra-clear tube</td>
 <td>Beckman Coulter</td>
 <td>344060</td>
 </tr>
 <tr>
 <td>SW60Ti ultra-clear tube</td>
 <td>Beckman Coulter</td>
 <td>344062</td>
 </tr>
 <tr>
 <td>SW40Ti rotor</td>
 <td>Beckman Coulter</td>
 <td>331302</td>
 </tr>
 <tr>
 <td>SW60Ti rotor</td>
 <td>Beckman Coulter</td>
 <td>335649</td>
 </tr>
 <tr>
 <td>Refractometer</td>
 <td>Fisher Scientific</td>
 <td>13-947</td>
 </tr>
 <tr>
 <td>Epifluorescence microscope</td>
 <td>Nikon</td>
 <td>Eclipse TE2000-E</td>
 </tr>
 </tbody>
</table>
Table 2. Reagents and equipment.

following cell-fate in e. coli after infection by phage lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Watch this Scientific Journal Video about Following Cell-fate in E. coli After Infection by Phage Lambda at JoVE.com

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

following-cell-fate-in-e-coli-after-infection-by-phage-lambda

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Immunology and Infection

22.9K visualizações.  University of Illinois at Urbana-Champaign. Este artigo descreve o procedimento para a preparação de uma versão marcada com fluorescência do bacteriófago lambda, infecção de E. coli Bactérias, após o resultado da infecção sob o microscópio, e análise dos resultados de infecção.