Ingegneria batteri aderenti mediante la creazione di un unico sintetico curli Operon

The overall aim of the following experiment is to give a adherent properties to bacteria in order to facilitate their separation from liquid media. This is achieved by first designing a synthetic curli on to bypass complex genetic regulation and enable easy and massive production of sticky polymers. As a second step, a adherent bacteria are visualized and quantified on polystyrene, which allows for a quick estimation of the efficiency of the synthetic device.

Next, a adherent bacteria are visualized on glass by confocal microscopy. In order to directly observe the biofilm and to gain information on its structure, results are obtained that show a significant increase of adherence in the strain harboring the synthetic device based on both adherence, percentage measurement and microscopic observations. The main advantage of the synthetic biology approach over existing methods like conventional cloning, is that construct harboring only the absolute clear required genes with appropriate signals can be very quickly obtained.

This method can help answer a key question in the bioremediation field, such as addressing the challenge of removing metal loaded bacteria from radioactive waste. Generally, individuals new to this method with struggle because basic knowledge about genetic regulatory signals and databases handling are required. Visual demonstration will focus on the quantification and visualizations of bio film because these two steps are very difficult to learn because of the fragility of bioframes.

The first step to designing the synthetic carron is to determine the genetic organization of the curly genes and to localize their endogenous, transcriptional and translational signals. This is accomplished using specialized databases such as REON DB or eco gene. The next step is to select the five absolutely required genes for curly synthesis, CSGB and CSGA and code the fiber monomers and C-S-G-E-C-S-G-F and CSGG encode the curling secretion complex, extract the chosen sequences from the database in faster format data management and in silico pre-analysis will be performed with clone manager software as the C-S-G-G-F-E cluster is antisense in the e coli genome.

This sequence needs to be converted into its reverse complement counterpart. Use the clone manager tools operations process molecule invert molecule, paste the C-S-G-E-F-G sequence behind C-S-G-B-A by using the function ligate. Next, add the appropriate promoter PRCN in front of the C-S-G-B-A-E-F-G iCal sequence.

The RCN locus encodes an FLX pump RCNA, responsible for nickel and cobalt detoxification, and its cognate metallo regulator RCNR. In the absence of cobalt or nickel, the repressor RCNR binds to the RCNR box on the DNA and prevents the full transcription activation of the downstream genes. If the intracellular concentration of cobalts or nickel rises, the binding of cobalt or nickel to the RCNR protein prevents the fixation of the repressor to the promoter and the transcriptional activity of P-R-C-N-A increases by placing the five selected curly genes under the control of the promoter.

PRCN curly are predicted to be overproduced in the presence of cobalt and nickel. Subsequently optimize the transcriptional signals by adding a perfect ribosome binding site or RBS in front of the first A TG of the CS gba, DNA sequence and a second perfect RBS in front of the C-S-G-E-F-G sequence. To fit I GMs standards, the device must be flanked by a standard bio brick prefix containing restriction sites for ECO R one and X-bar one, and a suffix containing restriction sites for SPE one and PST one to facilitate further assembly.

The bio brick part itself may not contain any ECO R one, PST one SPE one, and X-bar R one recognition sites. Hence, these sites were eliminated by silent mutation changes in silico restriction analysis of the device revealed one PST, one site in the CSGA sequence, one PST one site in the RCNR sequence and one ECO R one in the CSGE gene. These sites were modified by silent mutations.

Once the design of the synthetic opor is complete, run through a translation simulation using the clone manager software. Use the sequence alignment program blast to verify perfect homology between the wild type curly proteins and the proteins encoded by the synthetic on to begin this procedure for each well of a 24 well polystyrene plate with two milliliters of M 63, minimal medium with 0.2%glucose inoculate each well with 10 to the six cells of an overnight bacterial culture. Grow the bacteria at 30 degrees Celsius for 18 to 48 hours without shaking each column of the 24 well plate represents a modality.

The proper amount of cobalt and antibiotic is added when needed. The three first rows are used to quantify the A adherent bacteria by averaging the three repetitions. The last row left is used to visualize the biofilm by crystal violet staining after 18 to 48 hours, recover the supinate containing the planktonic cells from each well from the first three rows of the 24 well plate and transfer the supinate to a hemolysis tube.

Carefully rinse each well with one milliliter of M 63 medium and pull the wash in the hemolysis tube with the initial supinate. This pool is referred to as swimming cells named S.The bottom of the wells in the microplate is covered with adherence cells. To recover the biofilm, add one milliliter of M 63 into each.

Well scrape with the pipette tip and then pipette up and down and transfer the biofilm fraction to a new hemolysis tube. Vortex the tube for 15 seconds. Collect the biofilm fraction in hemolysis tubes.

This pool represents the surface attached cells named B transfer 200 microliters of the supinate and biofilm fraction from the hemolysis tubes to a 96 well plate. Estimate the number of surface attached and swimming bacteria from the optical density at 600 nanometers. The percentage of adherence for each modality is calculated by using the formula B times 100 divided by three times S plus B.To visualize the biofilm for only the last row of the 24, well plate discard the plankton cells from those wells.

Add one milliliter of M 63 to each well to rinse the biofilm that has developed on the bottom of the plate. Dry the uncovered plate for one hour at 80 degrees celsius after one hour, add 100 microliters of 20%crystal violet for two to each well followed by extensive washes with water. A fluorescent chassis is required for this protocol.

A suitable host for the plasmid bearing the synthetic curly on is the e coli SCC one strain, which constitutively express green fluorescent protein with no observable difference from the parent strain MG 1 16 55. Transform the host strain with a puck 57 plasmid bearing the synthetic curli to obtain the S 23 strain. Transform the host strain with a control plasmid puck 18 to obtain the control strain.

S 24 grow overnight pre cultures of the S 23 and S 24 strains at 30 degrees Celsius. In M 63 medium supplemented with glucose and ampicillin on the following day, add 20 milliliters of M 63 medium to a Petri dish and then inoculate 15 milliliters of M 63 in Petri dishes with 100 microliters of the pre cultures. Add the appropriate concentration of cobalt and don't forget the negative control without cobalt.

Introduce three rectangular glass cover slips into each Petri dish. Incubate the dishes overnight at 30 degrees Celsius without shaking. Bacteria can be visualized directly under the fluorescent microscope the following days.

It is important to avoid drying of the sample, so the following steps must be performed carefully but quickly. Remove each cover slip from the Petri dishes and carefully drain it. Carefully clean the lower face with a small cotton swab soaked with 70%ethanol to wipe off all bacterial residue for confocal observation.

Deposit the cover slip on a glass slide with the upper face covered by the biofilm placed face up. The biofilm is then covered with a larger cover slip, which is fixed to the glass. Slide with varnish.

Invert the setup so that the biofilm will now be on the lower face. Place the setup under the confocal laser microscope in this experiment. A Zeiss LSM five 10 meta confocal laser microscope is used within the CT micro platform.

Select the 40 times oil immersion objective. Use the following settings, excite GFP at 4 88 nanometers and collect the bacterial fluorescence in the range of 500 to 600 nanometers. Use the 40 times oil immersion objective for acquiring images.

In the laser scanning confocal mode. Scan the overall three dimensional structures of the biofilms from the solid surface to the interface with the growth medium. Using a step of one micron.

Perform three dimensional projections with a maus software, determine biofilm thickness by the analysis of the average C value along statistical longitudinal sections. Quantification of biofilm, bio volumes can be extracted from confocal Z stacks. The adherence abilities of e coli engineered to overproduce curly in response to metal can be assessed by simple methods.

For example, crystal violet staining on 24 well polished Irene plates allows rapid visualization of biofilm formation at the bottom of each well, as shown by the difference in purple color intensity. It appears that the wild type strain is less a adherent than the engineered strain. A quantitative method is to measure the percentages of adherence on polystyrene of the wild type and the engineered strains with various concentrations of cobalt.

The results presented in this graph reveal that the percentage adherence of the engineered strain is 1.5 fold higher than that of the wild type strain in the absence of cobalt. In addition, a significant reinforcement of adherence in the presence of increasing concentrations of cobalt was observed in media with cobalt concentrations of 25 micromolar or 50 micromolar percentages of adherence engineered cells reach 30%and 40%respectively. Adherence abilities can also be compared using microscopy with GFP tagged bacteria grown on glass slides, epi fluorescence microscopy observations of green bacteria on a black background reveal that the engineered strain forms a biofilm, whereas the wild trippe strain forms only micro colonies indicated by the arrows.

Confocal microscopy analysis provides an overall view of the biofilm three-dimensional structure. Top view reconstructions are shown in panels A and side view reconstructions are shown in panels B.The results confirm that the engineered strain is more a adherent and forms a denser and thicker biofilm than the wild type. While attempting this procedure, it is important to double check the operation design, for example, to explore bio mediation potential of modified e coli or occus ants in the context of radioactive wastewater.

After watching this video, you should have a good understanding of how to design a synthetic open using clone manager software and how to quickly quantify the utterance of bacterial strains. Don't forget that working with synthetic genes and genetically modified organisms can be extremely hazardous. They have to be under according to the current laws and precautions such as autoclaving transformed bacteria should always be taken while performing this procedure.