Expression Strength Of The ToxT and OmpT Promoters When Isolated
By: Samantha Bonge, Darlene Diaz, Charlotte Jenkins
Davidson College Honor Pledge:
“On our honor we have neither given nor received unauthorized information regarding this work, we have followed and will continue to observe all regulations regarding it, and we are unaware of any violation of the Honor Code by others.”
Pledged Sam Bonge
Our experiment was testing promoter sequences by inserting a promoter’s DNA sequence into a plasmid. We tested the expression by measuring red fluorescence (RFP) of the cell (if the oligo is not taken up at all, the cells will express with green fluorescent (GFP)). Our experiment had two phases: testing the ToxT promoter, and testing the OmpT promoter. Both promoter sequences are found naturally in Vibrio cholerae, and the ordered DNA sequences were tested in E. coli.
The first experiment tested whether the ToxT promoter is inducible or constitutive. Research has indicated that ToxT is amplified in the presence of the ToxR protein, so we tested the expression of ToxT without ToxR. We concluded that ToxT is in fact a constitutive promoter and can be expressed regardless of the presence of ToxR.
The second iteration of the experiment tested the OmpT promoter, which is linked to ToxT, but is repressed by ToxR. We hypothesized that OmpT should have stronger expression than ToxT. OmpT in fact had weaker expression than ToxT, which we suspect was due to the ordering limit of 60 base-pairs, which cut off the end of the DNA sequence for OmpT.
By using different promoters from cholera, we can gain a better understanding of how cholera can be combated as a whole. For the first experiment, since we would not have access to the ToxR protein, we decided to test whether or not expression of ToxT would occur without the presence of ToxR. ToxT promotes production of Cholera Toxin, and gives feedback to other promoters which aid in its secretion, as well as increased production of ToxR, creating positive feedback as more ToxR means greater expression of ToxT (Goss et. al).
Our research question for ToxT was: is ToxT inducible or constitutive? Research indicates that there is a region of the ToxT promoter that is ToxR non-inducible, although the expression in this region is weak (Goss et. al). In other words, the ToxT promoter region that we tested is not dependent on the presence of the ToxR protein to be activated and expressed; research showed expression increased with ToxR, but no trials were run without ToxR protein (Goss et. al). Our hypothesis was that ToxT is constitutive.
Fig. 1: ToxT sequence from Goss et. al.
The entire identified binding site from -104 to -56 was used.
Our second iteration involves the OmpT promoter. Our second research question was, in the absence of ToxR protein, is OmpT expression stronger than ToxT expression? According to prior research, OmpT is the only promoter in Cholera repressed by ToxR, which increases expression of the other promoters, including ToxT (Li et. al.). OmpT regulates the strength of the cell membrane; when OmpT is fully expressed, Cholera Toxin is not secreted because the membrane wall is strong enough to resist the secretion (Braude). When OmpT is repressed by ToxR, the membrane is weakened, allowing for lysing of the cell and the secretion of toxin (Li et. al.).
OmpT has higher expression than ToxT when without ToxR, as such, Cholera Toxin cannot be secreted. This is because ToxR induces the production the toxin, and represses the OmpT promoter while bolstering ToxT. So, in the absence of ToxR, ToxT should have very weak expression, and OmpT should have high expression, measured by RFP production in these experiments, rather than Cholera Toxin production.
Fig. 2: OmpT promoter sequence from Li et. al.
The truncated binding site from -92 to -37 was used. This region rather than the -95 to -30 region was used because of limitations of ordering sequences which could not exceed 60bp, including additional sticky ends.
The following methods are adapted from the Biology 113 Laboratory Manual.
First, we isolated the region we wanted to test, in this case, the ToxT and later OmpT promoters, and ordered the chosen DNA sequences. In order to test the expression of each promoter, each oligo was separately transformed and cloned using Golden Gate assembly (GGA) to be inserted into a plasmid, was was then taken up by E. coli cells. Cells used in this experiment had ampicillin resistance. The bacteria cells were plated with LB+Ampicillin, to prevent extraneous bacteria cells without our plasmid (which is also in the controls, minus the oligos we chose) from growing. Once colonies grew on these plates, liquid samples were made with three samples of experimental colonies, a negative control, and a positive control. The negative control went through GGA, but with water added instead of oligos, and should always express Green Fluorescent Protein (GFP). The positive control always expresses Red Fluorescent Protein (RFP). GGA is successful if the fluorescence expressed in the cells switches from GFP to RFP.
Samples of these liquid cell cultures were loaded into wells and analyzed with the Synergy machine. The Synergy machine measured the amount of RFP based on cell density in each sample. The greater the RFP, the greater the expression of the promoter.
In the first iteration of the experiment, the ToxT promoter samples were mixed with green master mix as the template to be used for PCR (Polymerase Chain Reaction), and we created a glycerol stock. PCR amplifies the DNA region inserted in the DNA, in order to verify the presence of the oligo in our experimental cells through gel electrophoresis.
For the second iteration of the experiment, PCR was not done with the OmpT promoter; instead minipreps were performed for the three experimental samples and the negative sample. The minipreps isolated the DNA for Sanger sequencing to confirm the presence of the oligo in the OmpT trial. This sequencing is more precise than the PCR from the first experiment.
The first promoter we tested was ToxT, to ascertain if ToxT was inducible (by ToxR) or constitutive.
Fig. 3: Experimental Plate with ToxT Promoter
The bright green spots are colonies that did not successfully uptake the oligo. The paler spots are pink and do express RFP.
Fig. 4: Fluorescence Strength of ToxT
Even without the other promoters cholera uses, and without the presence of the ToxR protein, ToxT showed weak but statistically significant expression. This shows ToxT is a constitutive promoter.
Fig. 5: Gel for ToxT samples.
From left to right: N, X3, X2, X1. The far right column contains the ladder.
To verify the presence of the promoter sequence in the cells, PCR was performed. The gel should have shown a defined band for the experimental groups at slightly below the bands for the negative control. Because this was not the case, the gel was inconclusive, though we suspect the promoter sequence is present because RFP was detected. There are a few possibilities why the PCR gel was inconclusive. The smearing out may be due to collecting a combination of cells with green and red expression. There may also have been nonspecific binding amplifying a similar part of the region leading to the large bands at the top.
The second promoter we tested is OmpT, which we expected to have strong expression, without the presence of ToxT.
Fig. 6: Experimental Plate with OmpT Promoter
The bright green spots are colonies that did not successfully uptake the oligo. The paler spots are visually pink, though the light box makes them hard to see in the photograph.
Fig. 7: Fluorescence Strength of OmpT
OmpT showed very weak, possibly statistically insignificant expression. OmpT had lower expression than the background expression of the negative control, though the negative control value was abnormally high; OmpT also had expression barely above that of the negative value from the ToxT experiment. OmpT had lower expression than ToxT, the opposite of what we predicted.
Fig. 8: This graph compares the expression of the two promoters.
Fig. 9: This is the same data as the above chart, with better resolution by omitting the positive controls. The negative control from the second experiment is abnormally high, and the OmpT expresses at about a third of the strength of ToxT.
The next figures show the DNA sequencing of the OmpT experiment. As expected, our experimental groups contain our oligo DNA sequence; the negative does not. We do not have the sequencing data for the Experimental 2, as the chromatograph produced by the Sanger sequencing was intelligible. There are about 60bp where the sequence of the Negative control does not match with the experimental groups, this is where the oligo is inserted in the experimental sequences, which is missing in the negative control (as it should be).
Fig. 10: Oligo Sequence vs. Experimental 1
*The asterisks show where the base-pairs match, showing where the oligo sequence is.
Fig. 11: Oligo Sequence vs. Experimental 3
*The asterisks show where the base-pairs match, showing where the oligo sequence is.
Fig. 12: Oligo Sequence vs. Negative
*There are no asterisks, meaning the oligo is not present.
Fig. 13: Negative vs Experimental 1 and 3
The first iteration of the experiment tested whether ToxT was a constitutive or inducible (by ToxR) promoter. ToxT showed significant expression of RFP, without the presence of ToxR which is not produced in E. coli, meaning the ToxT promoter is constitutive, supporting our hypothesis. PCR was meant to confirm the success of the DNA cloning but was inconclusive, though the weak but present expression of RFP supports the thought that it was successful.
The second iteration of the experiment tested if OmpT expression is stronger than ToxT expression without ToxR being present. ToxT seemed to be the main regulatory promoter of cholera, and we were curious if the other promoters linked to ToxT, of which OmpT is one, would express without the presence of ToxT. Because OmpT was the only promoter known to be repressed by ToxR, we chose OmpT for our second experiment.
We predicted that OmpT should express high levels of RFP. However, expression was very low, arguably negligible, meaning there is a possibility that OmpT is not a constitutive promoter, relying on the presence of ToxT or Cholera Toxin. While OmpT is repressed by ToxR and should express in its absence, it is possible OmpT is inducible by another molecule naturally found in Vibrio cholerae that was not apparent in our prior research. The other possibility for such low expression is that, because of the limitation of only ordering a sequence of 60 base-pairs, several nucleotides on either side of the promoter had to be omitted, which could have altered or prevented its function. Through DNA Sanger sequencing we confirmed the presence of our promoter in the cells, so the lack of expression was not a result of failed cloning.
The positive and negative control values were expected to remain about the same between the two experiments. While this is true for the positive value, the negative value was much higher than expected in the second. It is unclear what caused this, but the negative control from the OmpT experiment should be disregarded when judging expression of OmpT.
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