Team:British Columbia/Results
Table of Contents
Pre-experiments
OD growth curve
Screening the Pre-existing E.coli Library
We screened around ~2300 different promoters from an E. coli promoter library. Using the Labcyte Echo, the dispensing and fluorescence readings were done mechanically, over 24 hours. Over 4 rounds of screening, we managed to narrow down the library to 7 leads. The analysis was done by selected outliers that were 3 adjusted standard deviations from the median. Looking at the most recent screen, each data point was adjusted to the control (GFPsample/GFPcontrol), and normalized to 1 OD600 ensure accuracy and representation. Each sample had 3 replicates, and, only one 1 out of the 7 leads (row 4, column 18) had a more consistent spread across the 3 replicates. Additional tests, such as more replicates and sequencing, needs to be done to fully ascertain whether these do have the potential to be made into a robust biosensor for saxitoxin.
We can conclude that most likely not cell’s intrinsic mechanisms acting on the saxitoxin.
Supplementary Data Analysis Files
SIGEX
Transformation data
For SIGEX, we transformed around 100,000 different clones, ligating our vector plasmid with the prepped metagenomic library. Using vector ligation controls, we determined that there were negligible amount of self-ligation vectors.
Supplementary Transformation data
[1] The pictures of the plates of our transformation can be found here
Cell Sorting
After pooling all our clones, we used the Chipbio cell sorter to sort out our cells. Alongside saxitoxin, we screened biphenyl and P-coumaric acid, two common effector molecules (source) found in the Saanich inlet (where we acquired our DNA). These compounds were tested as a way to confirm the efficacy of our method. If known promoters were found, it would be another way to prove our experiments worked.
Initially sorter sorts through 1 million cells from the pooled DNA library, removing the constitutively fluorescing cells above a certain threshold. As seen in the image below, 93,958 cells were kept while the others were sorted out.
In the second round of sorting, the cells were incubated in their respective effector compound before being sorted. In the saxitoxin sort, 2,741 cells expressed relatively strong fluorescence and was kept for further testing.
Efficacy Test
After sorting the cells, we plated the cells and inoculated 96 well plates with the colonies. When the cells were grown up, we transferred them into a 384 well plate with their respective compounds. A screen was performed, and out of the many candidates a total of 9 different clones were chosen for the final tests. The experiment we ran was a titration experiment, with 5 different concentrations, over a time of 16 hours. The results of the test is shown below in the figures. Overall, many of the samples indicate a favourable increasing trend of increasing GFP expression when STX concentration increases. The 0.05ppm STX is seen to not be a high enough concentration to induce any increased relative fluorescence, with most responses starting at 0.4 ppm over some time. Some samples, such as sample 3, have surprising aberrations that may be due to experimental results, but more testing will be needed to ascertain the causes.
Data Analysis
[Note:Bar graph: All raw data was first normalized to 1 OD600. The bar graph shows all the relative fluorescence of each sample. Sample 2 and sample 9 did not grow or fluoresce and therefore not shown here. Rejected samples, such as replicate 1 for 2.0ppm 2ppm for all samples, were removed for that same reason. The data here is selected for the best overall spread at a time point (This is not necessarily indicative of strongest fluorescence expression. It is just the time point where the overall trend is easiest to visualize.)]
[Note2:Line graph: The data points of each well were divided by their control to obtain their fold increase compared to the non-fluorescing control. As seen, the control is located at 1 at the x axis; anything below 1 has less relative fluorescence compared to the control, and anything above 1 has more. ]
Since there did not seem to be any information about saxitoxin’s effect on E. coli growth, we needed to run some experiments to confirm this for ourselves. We ran a HCl solution control as the saxitoxin itself was stored in 3 mM HCl. The results seen from the experiments showed that saxitoxin itself did not affect the growth curves of the E. coli, matching the growth curves of the control.
Supplementary Data Used in Analysis
Conclusion
From over 100,000 different clones and millions of cells sorted, we finally narrowed it down to a handful of potential candidates for our biosensor. The next steps will be to run additional tests on these candidates to fully confirm whether they have to potential to be made into biosensors. Currently we are sequencing the lead hits we tested out, which unfortunately will not be done before the wiki freeze. If we do confirm our hits, we can move on to optimizing these inducible promoters into transcription-based biosensors for downstream applications.