Team:Guelph/Results

Results

This page represents our highlighted and core experiments that allowed iGEM Guelph to produce our AmilCP and Violacein Biosensors. This is not an exhaustive list or presentation of the work done. For any further clarification, please refer to our lab notebook as all of our experiments are catalogued there. In order to create our biosensor, we began by setting out to determine whether the common lab Escherichia coli strains - DH5α and BL21(DE3) - would be able to survive high enough concentrations of tetracycline to be useful as a biosensor. This was done by performing minimum inhibitory concentration (MIC) assays in a 96-well plate. We also mapped a growth curve of both strains in the presence of tetracycline in order to see what quantities of the drug inhibited growth.

Initial Work with E. coli in Tetracycline

To test the tolerance of E. coli to tetracycline, DH5α and BL21 (DE3) strains were grown in a 96-well plate, each in 200 μL of LB, in serially diluted tetracycline from 40 μg/mL to 0.04 ng/mL. The plates were shaken at 900 rpm at 37 °C, and the optical density at 600nm was recorded after 24hours using the Tecan Infinite Series Pro 200 96-well plate reader.

When the ODs were normalized to the no drug control, we were able to calculate our MIC₅₀, which is the value of where the growth of the cells are inhibited by 50%. We saw that our MIC₅₀ was at 312.5ng/mL as described where the normalized OD values fell below 0.5. This means that half of the cells from both strains were able to survive in up to 312.5 ng/mL tetracycline compared to cells growing in wells without tetracycline. This was great news as 312.5ng/mL is slightly more than three times the regulatory limit for tetracycline in dairy products in Canada (100 ng/mL). With this information, we felt confident that both DH5α and BL21 (DE3) strains would survive above the regulatory limit and will be used for the development and initial testing of our tetracycline biosensor.

Now that we know our strains were fine, we ordered the plasmid pJKR-H-TetR (p-Tet) from addgene that is the backbone on which we based our tet-O DNA synthesis¹. We then needed to test the rate of growth of our strains in ampicillin and tetracycline as ampicillin will be needed to maintain the plasmid. We transformed our pTet plasmid that contained ampicillin resistance into our DH5α strain. We then created growth curves by growing E. coli in LB broth at 37°C shaking at 250 rpm overnight. The cultures were then diluted 1:1000 in 50 mL LB media. Based on the paper we obtained p-Tet from, the highest dose of tetracycline the researchers used was 267ng/mL. We wanted this experiment to quantify the impact that tetracycline would have on growth and capture the start of log phase for these cells. In our future work, we will be using lower volumes of tetracycline; So we wanted to capture growth in the highest concentration of tetracycline (and therefore the longest lag phase). So for this experiment we added tetracycline to a final concentration of 267 ng/mL and ampicillin to a final concentration of 100 μg/mL. For our negative control, we diluted the cultures into flasks that contained 100 μg/mL of Amp, without any tetracycline. The flasks were incubated shaking at 250 rpm in 37°C for 14 hours, taking absorbance readings at 600 nm every 2 hours.

From the analysis of our growth curves, we were able to determine that E. coli in tetracycline begins log phase after 4h of growth. Our control strain had almost no lag phase and started logarithmic growth almost immediately. This means that after 4 hours, with the highest concentration of tetracycline that we would use to induced expression of our pigments, we should clearly see colour being produced. We anticipate based on these results that the longest time is should take the cells to reach log phase would be 4 hours and with lower concentrations of tetracycline, it should take even less time. This information was used when determining when to harvest cells for expression testing.

Validating the Responsiveness of BBa_K3189001 to Tetracycline

The next step was to determine the sensitivity of our new biobrick, BBa_K3189001, a tetracycline-activated phage lambda promoter. To do this, DH5α and BL21(DE3) cells containing the pTet plasmid were grown in 50 mL LB media containing tetracycline to a final concentration of 50 ng/mL, as well as 100 μg/mL ampicillin to maintain the plasmid. We chose 50 ng/mL as it was under the regulatory limit and we wanted to push the limits of our test so we also measured 1ng/mL which previously showed no additional fluorescence beyond background levels (data not shown). The flasks were incubated and shaken at 37°C at 250 rpm for 6 hours. The 6 hour time point was chosen based on the point where we were confident that the cells were past log phase from our previous growth curve experiments. After six hours, the cells grown in flasks were washed and resuspended in PBS to reduce the autofluorescence by LB media. When assessed under UV light, it was clear that the 50 ng/mL of tetracycline showed a visible increase in fluorescence compared to the same cells grown in only 1 ng/mL tetracycline (Figure 3).

Since GFP was visibly produced with tetracycline present at only 50 ng/mL, half the maximum concentration of tetracycline allowed in dairy products in Canada, we concluded that BBa_K3189001 is sensitive enough to tetracycline to be useful in our biosensor.

E. coli BL21 and DH5α cells containing pTet were also grown in a 96-well plate with a decreasing gradient of tetracycline in the same manner as a MIC plate. We did this attempting to establish a linear relationship between concentration of tetracycline and fluorescence by growing pTet containing BL21(DE3) and DH5α cells in sequentially diluted concentrations of tetracycline in a 96-well plate. Unfortunately, we had difficulties with our Tecan Infinite Pro200 plate reader measuring fluorescence, so we were not able to establish a quantitative relationship between the two variables. However, we were able to visualize the 96-well plate under UV light to qualitatively assess this relationship (Figure 4).

What we observed was that as expected, fluorescence generally increased with higher tetracycline concentrations. The brightest fluorescence was seen at 312.5 ng/mL highlighted in red. This was interesting as this is the same value that is the determined MIC₅₀ of E. coli in tetracycline) This may be due to tetracycline affecting the cell’s metabolism and above their MIC50 that they grow so poorly or no longer have a functioning metabolism that allows them to adequately respond to the tetracycline by producing GFP.

Next: Demonstrate

University of Guelph iGEM 2019