Part:BBa_M50116:Experience

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Methodology with Results

Growing Bacteria

Our plasmid construct was provided by DNA 2.0 in powdered form; we reconstituted the plasmid in sterile water following DNA 2.0’s instructions. We transformed E. coli cells as described in the transformation protocol of Practical 3 in the BioE44 Course Reader and Lab Manual. The cells used were prepared earlier in the course also following the protocol from Practical 3. The transformed E. coli containing pTMAO were then grown on LB + ampicillin (amp) agar plates. This allowed for selection of the bacteria transformed with pTMAO due to the amp resistance in our plasmid. Following the transformation, we picked single colonies and grew them up overnight at 37oC shaking in LB + amp for all experiments, unless otherwise indicated. From this liquid culture, we created a glycerol freezer stock for the plasmid. For this step, we followed the protocol outlined in Practical 4 of the BioE44 Lab Protocol Booklet, and stored our stocks in the -80°C freezer (barcodes 0133021751 and 0133027053).

Overnight Dose Dependent GFP Assay

In order to test the initial dynamic response of expression from the torC promoter to different concentrations of TMAO, we used the the E. coli transformed with pTMAO grown in media with different concentrations of TMAO and measured the Green Fluorescence Protein (GFP) fluorescence. We grew the transformed bacteria in EZ rich defined media with glycerol and amp containing the following concentrations of TMAO: 2.5, 5, 10, 20, 40, 80 mM. We based our initial concentration range of this GFP assay off of which used 40 mM TMAO to conduct their experiments. For our positive control, we used a dose of 200 mM TMAO since this is a much greater concentration than all of our samples. Our negative control contained the transformed E. coli and EZ rich defined media, but did not contain any TMAO. All samples were initially prepared to 0.2 mL total volume in a 96-well deep well plate in triplicate and the OD600 and fluorescence (400 nm/515 nm). The samples were covered with a breathable membrane and were read every 30 minutes beginning at 7:55 pm May 21st to characterize a 12 hour period after TMAO induction. All samples had a starting OD600 of 0.01.

GFP Fluorescence at 12 hours after TMAO dosing within a continuous plate read experiment. Error bars represent standard deviation. This trend of leveling out towards 80 and 200 mM of TMAO indicated that we should expand the lower limit of our dose curve in order to fully capture the range of sensitivity of this composite part as well as expand the time frame used to characterize the difference in response between concentrations.

Longterm Dose Dependent GFP Assay

In order to test the concentration of TMAO sufficient to fully induce expression from the torC promoter, we used the the E. coli transformed with pTMAO (BBa_M50116) grown in media with different concentrations of TMAO and measured the Green Fluorescence Protein (GFP) fluorescence. We grew the transformed bacteria in EZ rich defined media with glycerol and amp containing the following concentrations of TMAO: 2.5, 5, 10, 20, 40, 80 mM. We based our initial concentration range of this GFP assay off of which used 40 mM TMAO to conduct their experiments. For our positive control, we used a dose of 160 mM TMAO since this is a much greater concentration than all of our samples. Our negative control contained the transformed E. coli and EZ rich defined media, but did not contain any TMAO. All samples were initially prepared to 1 mL total volume in a 96-well deep well plate in triplicate and kept in a shaking microplate incubator at 37oC and 750 rpm. All samples has a starting OD600 of 0.01. In order to test how long it took our promoter to express GFP in the presence of TMAO, we decided to use three time points for all of our readings, which were 12, 24, and 48 hours after TMAO induction.

At each of the indicated time points, 200 uL of each sample was transferred to a clear 96-well plate to be measure the GFP fluorescence and the cell density of each sample using the plate reader. We used 200 uL of EZ rich defined media with glycerol and amp as our blank for the plate readings. The cell density was measured by measure the absorbance at 600 nm (OD600). The GFP fluorescence was read at the GFP excitation wavelengths of 400 nm and an emission wavelength of 515 nm.

For the first trial, we used a gain of 100 in order to read our fluorescence values. Thus, at the 24 and 48 hour time point readings, we obtained overflow values for our fluorescence reading. We were able to obtain a 12 hour reading using a gain of 100 for our fluorescent measurement. For the rest of the dose dependent GFP assay experiments, we read the fluorescence values using a gain of 50 to accommodate for higher levels of GFP produced over time. From our results of the 12 hour time point, we were able to get a top limit to our dosing curve but were unable to view a bottom limit and so we changed our dosing concentrations to 0.625, 1.25, 2.5, 5, 10, 20, 40, 80 mM. The positive and negative controls remained the same and this range of TMAO concentrations was used for the rest of the drug-dosing GFP assay experiments.

Trials 4 and 5 GFP Fluorescence at 12, 24, and 48 hours after TMAO dosing. Error bars represent standard deviation. The negative controls are not included due to log2 scaling of x axis for readability of lower concentrations, but on the whole the 0mM wells produced less GFP than wells with TMAO added. The exception to this would be the 80 mM samples in trial 5.1, marking these samples as outliers. There did not appear to be a consistent dose curve or indication of a maximum, even over 48 hours, and thus we decided to do a final trial with cells from a different plate and read GFP florescence at 12 and 24 hours.

Trial 6 GFP Fluorescence at 12 and 24 hours after TMAO dosing. Error bars represent standard deviation. The negative controls are not included due to log2 scaling of x axis for readability of lower concentrations, but was lower than 1.25 mM in the 12 hour read and .625 mM in the 24 hour read. Concentrations lower than 10 mM in the 24 hour read appear to be equivalent while the positive control (160 mM) is smaller than the fluorescence of the 10 mM response. This could be due to the intake and reduction of TMAO to TMA reducing the number of molecules to sense and increasing the stress from the following alkaline conditions.

This experiment was repeated for 5 trials more for a total of 6 trials. While the initial results from the first trial indicated that 0.625 mM to 160 mM of TMAO would be in range over 24 hours in EZ rich defined media, there is not a clear dosing curve from these results. We will only report the data from the last 3 trials as they had the cleanest execution and data collection of the 6 trials. The second trial’s plate read was not performed with a blank and the third trial was performed with inadequate volume, rendering their data either incomparable or inaccurate. The 5th trial included 2 sub trials within the same 96 well plate to increase the amount of replicates and data available to compare. Due to time lab hour constraints, the 5th trial’s plate read was performed at times within 2 hours of 12, 24, and 48 hours, but not exactly on the mark. Finally, the 6th repeat trial was performed using a colony from another plate, in order to confirm fully the trend we were seeing across multiple plated colonies.

Anaerobic vs Aerobic Growth Condition GFP Assay

Using the same GFP assay as above, we tested to see how GFP fluorescence differed when our transformed E. coli was in anaerobic and aerobic conditions. For this experiment, we used a concentration of 60 uM TMAO, since this was found to be a sufficient amount of TMAO to induce expression of from the torC promoter. Our positive control contained 120 uM TMAO and our negative control had no TMAO. The aerobic conditioned was prepared as described in the dose dependent GFP assay above. The anaerobic condition was created by using 1.5 mL eppendorf tubes. We had the same TMAO concentrations as described for the aerobic condition. All tubes began with a OD600 of 0.01. We made three tubes for each condition in order to test the conditions at three different time points: 12, 24, and 48 hours. The tubes were filled to 1.5 mL total with EZ rich defined media. This was to prevent as little air exchange as possible. Before each sample was added to the eppendorf tube, the tube was flushed with nitrogen (N2) gas to eliminate or limit the oxygen content initially present in the tube. The tube was then closed and parafilm was used to seal the top of the tubes. Each tube was then placed in a rack and put into the 37oC incubator.

pH Controlled Media GFP Assay

Using the same GFP assay as described above, we tested to see how GFP fluorescence differed when our transformed E. Coli was grown in a MOPs buffer solution. Since the EZ rich defined media already contained MOPs, we decided to use LB + amp media for this experiment. To make the media for this experiment, we added 5 uL of 1 M MOPs and diluted it to 50 mL with LB + amp media to get a final concentration of 100 mM MOPs. We then used 1 M HCl acid to titrate the LB + amp + MOPs media back to a pH of 7, which is the optimal pH for E. coli growth. We then has the same positive control, negative control, and sample concentration as described in the anaerobic/ aerobic GFP assay method. We prepared the MOPs buffered samples for both aerobic and anaerobic conditions as described in the anaerobic/ aerobic GFP assay method.

GFP Fluorescence in Anaerobic vs Aerobic and pH controlled Media at 12 and 24 hours after TMAO dosing. Error bars represent standard deviation. No LB normalization due to error with blank. Anaerobic pH controlled conditions produced promising increase in GFP production that should be optimized and repeated in the future

For future experiments we recommend validating optimal conditions within anaerobic, pH controlled media within a 12 or 24 hour time frame to produce an optimized dose response curve. However it should be noted that our lack of a dose curve and the decrease in GFP fluorescence within higher samples in some cases may be due to stress from the reduction of TMAO to TMA, which would reduce the amount of TMAO available to sense on longer time scales and produce stressful alkaline conditions that may interfere with the activation of the protein torR and thus our plasmid.

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