We conducted an experiment to compare biofilm production between wildtype E. coli and E. coli containing our Csg construct. We visualized the biofilm production rate through crystal violet staining.

1. Inject diluted LB liquid cultures of E. coli into a 96-well plate
2. Cover the plate with the lid supplied by the manufacturer
3. Incubate the microtiter plate for 24 hours at 37°C wrapped in a plastic bag with almost no air left in it.
1. The lack of oxygen should put a small level of stress on the E. coli, inducing it to produce biofilm.
4. At the 12 hour mark, inject another set of LB liquid diluted cultures of E. coli into the 96-well plate.
5. After incubation, extract the liquid containing the bacteria into an eppendorf tube, leaving the biofilm on the well wall, via pipette for later culturing.
1. It is impossible to extract all E. coli from the 96-well plate without disturbing the biofilm as the biofilm serves as a platform that adheres the bacteria to the biofilm complex, and even after extracting the liquid containing bacteria there would still be a portion of bacteria left on the biofilm. However, extracting the bacteria would ensure a higher level of accuracy in the ELISA Reader readings, and the tiny amount of bacteria left on the biofilm should not affect the results and data of the ELSIA Reader.
2. We also made sure that there were no foreign species bacteria in our 96-well plate through the use of Chloramphenicol antibiotics injected into the LB broth.
6. After the extraction of bacteria, inject three cultures of E. coli into three wells and stain them with 0.1% crystal violet.Wear gloves and a lab coat while making the solution. Use caution when weighing out the Crystal Violet as the powder is hygroscopic and readily stains clothing, skin, etc.
7. After 15 minutes, extract the all the liquids from the three wells
1. These 3 wells are a blank that show what E. coli looks like when stained by crystal violet, and its effect on any E. coli left on the biofilm after the E. coli has been extracted.
8. Add 125 μL of a 0.1% solution of crystal violet in water to the other wells of the microtiter plate.
9. Incubate the microtiter plate at room temperature for 10-15 min.
10. For qualitative assays, the wells can be photographed when dry.
11. Add 125 μL of 30% acetic acid in water to each well of the microtiter plate to solubilize the CV.
12. Incubate the microtiter plate at room temperature for 10-15 min.
13. Transfer 125 μL of the solubilized CV to a new flat-bottomed microtiter dish.
14. Quantify absorbance in a ELISA plate reader at 550 nm using 30% acetic acid in water as the blank.
15. Extract 1uL of bacteria from the eppendorf tube
16. Dilute the bacteria to 1000x in LB broth and culture it in LB agar in a petri dish at 37°C for 12 hours. Count the colonies grown afterwards to observe growth rates

The crystal violet stain was used to stain the first three wells of the 96-well plate as a blank, and some pretests at very low dilutoins for the functional assay were performed in the three rows of wells below. The final four rows were the final and official functional assay performed from where we got our data. The wells were stained a dark indigo from the crystal violet on the biofilm, and the presence of biofilm was confirmed through the prodding of the well walls with a pipette, feeling a thick gooey snot-like substance.

From the final average concentrations calculated from the optical density values read from the ELISA Reader, we can clearly see distinguished differences in the amount of biofilm between the wild type E. coli and engineered E. coli. The Engineered E. coli had a higher concentration of biofilm after both 12 and 24 hours of incubation, proving the success of our design in curli fiber production.

We went further to investigate the effects of the plasmids on the growth of bacteria in case the Csg plasmids exert too much stress on the E. coli and hinder bacterial growth. After incubating the bacteria in the 96 well plate for biofilm growth, we extracted the bacteria grown from the well plates, and used a fixed amount of bacteria to culture in petri dishes.

We diluted the culture to 1000x and cultured it at 37°C in LB Agar, and distributed the bacteria into four different plates. A plate that grew wild type E. coli for 12 hours, a plate that grew engineered E. coli for 12 hours, a plate that grew wild type E. coli for 24 hours,and a plate that grew engineered E. coli for 24 hours. After 12 hours of growth the wild type E. coli plate yielded only 2 single colonies, while the engineered E. coli plate yielded 91 colonies. After 24 hours of growth, the wild type E. coli plate yielded 451 colonies and the engineered E. coli plate yielded 496 colonies. These numbers suggest that our plasmid does not hinder the growth of E. coli, and grows just as well if not better than the wild type E. coli.

Although the data collected in the Biofilm assay implies increased biofilm growth with the addition of our construct, there are multiple points of imperfection that could be improved.

Firsty, we foresaw a problem that may occur in the collection of data in the ELISA reader. Since biofilm is a adhesive platform that will stick to bacteria, it is impossible to remove all the bacteria from the 96-well plate, and the different amount of growth of each group of bacteria could cause an inaccuracy in the readings. To grasp just how much inaccuracy the bacteria could cause, we made our three blank wells to visually view how much the E. coli would effect the data collected. As it turns out, there was not much visible staining that occurred and this implies that the presence of E. coli was very little and barely observable, and does not affect the outcome of our data. The petri dishes we cultured from the bacteria in the 96-plate well also illustrates the difference in the growth rate of each group of bacteria, and from the resulting number of colonies we can see how the growth rates between wild type and engineered E. coli differ only slightly, and the growth rate should not be a problematic factor that hinders the outcome of our data.

Moreover, although our construct yields more biofilm than the wild-type E. coli DH5Alpha, the amount of biofilm produced after 24 hours is too little to be of effective use in environments such as the ocean. From literature, we see that E. coli MG1655 produces low amounts of biofilm due to downregulation by CsrA RNA-binding protein, we plan to investigate the genetic makeup of SAR11 in the future in order to eliminate similar biofilm downregulation mechanisms. By knocking out CsrA or similar genes that downregulate biofilm production through the binding of sRNAs that plays a major role in controlling biofilm formation, we hope to shorten biofilm growth time and boost production.

Another issue in the data we collected was the anomaly of the 2 colonies grown in the plate of wild type E. coli cultured for 12 hours. After 12 hours of incubation, there should be more than 2 colonies of bacteria grown in the petri dish. The inaccuracy here is probably caused by human error, as issues such as pippetting errors and the pollution of foreign antiobitics in the culture could be the cause of such a little bacterial growth in the culture.

We conducted an assay to confirm that our MetJ construct effectively suppresses biofilm production at high bacterial population densities. We grew multiple cultures of E. coli containing both the MetJ fragment 3 and our Csg construct. Using an ELISA reader and various staining methods, we measured population density and biofilm levels.

Purpose: To prove a decrease in biofilm growth at high cell densities

1. Grow a culture of the engineered E. coli (Containing Csg construct & MetJ Fragment 3).
2. Place multiple cultures of engineered E. coli (Containing MetJ Fragment 3 and Csg construct) in 21 tubes.
1. Extract in 7 different time intervals.
2. Test for crystal violet staining.
3. Repeat 3 times for each time interval.
3. Dilute the culture 1:100 into LB broth.
4. Add 100 μL of the dilution per well in a 96-well dish. For quantitative assays, we typically use 4-8 replicate wells for each treatment.
5. Incubate at 37°C.
6. Extract 6 test tubes every 2 hours for 14 hours.
7. Measure live population through flow cytometry and crystal violet staining.
1. To quantify E. coli population and the amount of biofilm.

We failed to co-transform our Csg construct and our MetJ 3 fragment into E. coli. For future testing, we will proceed with the MetJ Assay.

The failure for the co-transformation of our Csg construct and our MetJ 3 fragment into E. coli is probably caused by the difficulty to transform two different plasmids into a single bacteria. The presence of two new plasmids would put a very high amount of stress on the bacteria, and the bacteria would sometimes choose to eject the plasmid out its body to relieve such stress. This makes it hard for us to transform both our Csg and MetJ plasmids into the E. coli.

To overcome this obstacle, we can utilize a different approach to transforming our gene fragments into E. coli. We could utilize Gibson assembly to combine the MetJ and Csg fragments into one plasmid. Although this plasmid would be of a larger size, it would be easier to transform a single alrge plasmid into a bacteria than gambling to transform two moderately sized plasmids into a bacteria, hoping that it will accept both plasmids. This is a very achievable process, but due to time constraints, we could not implement this method to carry out the MetJ Protein functional assay.

Our goal is to illustrate the survival pattern of our engineered E. coli bacteria, showing that they perish at low culture concentrations but thrive at high concentrations. To achieve this, we will assess the growth of E. coli containing our biosafety plasmid at different liquid culture concentrations. This assessment not only helps us evaluate the efficacy of our safety mechanism but also identifies the threshold at which bacterial population decline begins, offering insights for future project optimizations.

1. Incubate liquid culture of E. coli with biosafety plasmid in a liquid culture and incubate overnight at 37°C, shaking it at 250 rpm
2. Make subcultures inside a 96-well plate and incubate overnight at 37°C
3. Measure the population concentration of the culture using an ELISA Reader
1. Stain the well cultures with SYTO 9
2. Use an ELISA reader to measure the optical density at a wavelength of 483
4. Using LB broth, dilute the existing culture to multiple diifferent liquid concentrations with 3 cultures of each concentration
5. Using the quantity of culture used, calculate the starting concentration of E. coli
6. Incubate the cultures for 12 hours at 37°C, shaking it at 250 rpm
7. Subculture these orignial cultures in a 96-well plate overnight for 12 hours at 37°C
8. Stain the subcultures with both SYTO 9 and PI stains, which can indicate both live and dead cells in a culture
9. Using flow cytometry, measure the ending concentrations of the SYTO 9 and PI stains, with red indicating PI, which stain dead cells, and green indicating SYTO 9, which stian live cells.

If our biosafety mechanism works, then the wells with low concentrations of bacteria should have more red stains than green ones, and wells with high concentrations of bacteria should have more green stains than red ones. More red stains would indicate that the toxin, MazF, is overwhelming the antitoxin, MazE, and vice versa. This would mean that our quorum sensing based biosafety mechanism is functional and efficient. One piece of data we can get from the functional assay is the threshold of the bacterial population density at which the toxin starts overwhelm the antitoxin.

In order to preempt potential problems that may aries, we have outlined problems that we may encounter and devised specific improvements to our design to address them.

1. E. coli growth does not decrease at low concentrations
1. Reduce anti-toxin expression: The constant anti-toxin production may be too high, neutralizing the toxins produced even at low bacteria concentrations. We will use a weaker promoter for anti-toxin gene expression. The optimized strength of promoter can determined by biosafety model
2. E. coli growth plummets, bacteria dies even at high liquid culture concentrations. This may be caused by (1) an overwhelming amount of toxins that remain under high bacteria concentrations that the anti-toxin cannot fully neutralize, resulting in bacteria death. (2) The quorum sensing population threshold to trigger repression in toxin production is too high, causing the bacteria to die even under concentrated populations.
1. Increase anti-toxin production to neutralize remaining toxins in the bacteria under high bacteria concentrations.
2. Change quorum sensing molecule to have a lower population threshold to trigger toxin repression under lower bacteria concentrations.

We aim to determine the reaction constant between the toxin and antitoxin through quantifying toxin produced by expression a gene that encodes for Green Flourescent Protein (GFP) alongside the MazF protein. By quantifying toxin production alongside bacteria population change, we can calculate the reaction constant for toxin and antitoxin. This allows us to complete the rest of the Biosafety Model (link to biosafety model), to determine the ideal antitoxin production rate to be able to limit the bacteria population exactly where we want it to be.

We aim to prove a decrease of individual bacteria biofilm growth rate at high cell densities. We plan on growing multiple cultures of engineered E. coli (with MetJ and Biofilm plasmids) for different amounts of time, and measuring their bacteria population and amount of biofilm through ELISA reader with different staining methods. This allows us to see the change in bacteria population and amount of biofilm over time, which will allow us to calculate the individual bacteria biofilm production rate overtime. We expect to see an overall decrease in individual bacteria biofilm production rate as bacteria population increases.

1. Put multiple cultures of engineered E. coli (with MetJ and Biofilm plasmids) in multiple test tubes 6x7, 42 tubes, 7 extracts, 3 tubes for SYTO9 and PI (live and dead) cell stains, 3 tubes for crystal violet staining (measure biofilm).
2. Dilute the culture 1:100 into LB broth for biofilm assays.
3. Add 100 μL of the dilution per well in a 96-well dish. For quantitative assays, we typically use 4-8 replicate wells for each treatment
4. Incubate at 37°C
5. Extract 6 every 2 hours for the duration of 14 hours
6. Measure live population through ELISA reader with SYTO9 and PI (live and dead) cell stains
7. Measure amount of biofilm through ELISA reader with crystal violet stains
1. Allows us to quantify E. coli population and the amount of biofilm using the population and biofilm measurements, we can calculate the bacteria individual biofilm production rate over time.

In order to preempt potential problems that may arise, we have outlined problems that we may encounter and devised specific improvements to our design to address them.

1. No biofilm / low amounts of biofilm is produced.
   This means that our MetJ construct is too potent, producing too much MetJ proteins to repress biofilm production. To resolve this issue, we will:
1. Weaker promoter: The promoter for our MetJ construct may be too strong, we will replace it with a weaker promoter.
2. Change Quorum Sensing molecule: Different quorum sensing molecules have different thresholds of concentration that is required to trigger a response in the bacteria, by adjusting our quorum sensing gene to produce molecules that require a higher threshold to trigger MetJ protein production, we can prolong biofilm gene expression.
2. Biofilm production rate increases
   This means that our MetJ construct does not function, or may be too weak.
1. Stronger promoter: Promoter for MetJ construct may be too weak, decreasing the construct’s effectiveness in regulating biofilm gene expression, we will replace it with a stronger promoter.
2. Change Quorum Sensing molecule: Change our quorum sensing molecule to require a lower threshold in triggering MetJ protein production.
3. Weaker biofilm promoter: Our biofilm gene expression may be too strong, cancelling out the effects of our MetJ construct in repressing biofilm growth. By adjusting our Csg construct promoter to a weaker alternative may magnify the effects of the MetJ construct.