Team:OhioState/Results

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Results

Promoters

For our contribution, we tested 2 individual promoters, pFraB (BBa_K3783000) and pR (BBa_R0051), and 1 fusion promoter, pR-pFraB (pC) (BBa_K3783001). We coupled these promoters to a luciferase reporter plasmid pSB401 in fraR positive and fraR negative strains of E. coli. They were tested with and without IPTG due to its upregulation of fraR. All of the luminescence data was normalized to the OD across 20 hours.


FraR

The fraR+ strains worked as intended, by repressing the expression of luciferase compared to the fraR- strains (their relative light units are the lowest in figure 1). The presence of IPTG should affect the luminescence because fraR is expressed by a lacZ promoter, however there was only a slight difference in the two trials.

pFraB Graph
Figure 1. pFraB Strength Measured by Luminescence

Controls

We ran two controls. The pR promoter by itself served as a positive control and provided more characterization for an existing part. An empty pSB401 vector, served as the negative control. The pR worked as expected; it had a strong expression of protein regardless of the presence of IPTG. Seen in figure 2, both of the pR series have high relative light units no matter if IPTG was present. The negative control also produced expected results with negligible luminescence produced. We submitted this luminescence data of pR to the biobrick registry in order to provide more characterization data of this promoter to future iGEM teams.

Control Promoter Graph
Figure 2. Control Promoter Strength Measured by Luminescence

Fusion Promoter

For our fusion promoter, a noticeable decrease- about threefold- was observed between the fraR+ and fraR- strains, as expected. The presence and absence of IPTG seems to be trivial although its effects can be seen in figure 3 and are consistent. The fraR- strain with IPTG had the highest levels of expression, and although they should hypothetically have the same expression, fraR- with IPTG expresses slightly less. The fraR+ strains do show a significant decrease though, with the IPTG trial being slightly more repressed than the trial without IPTG. All of this follows our hypothesized function.

pR-pFraB Graph
Figure 3. Fusion Promoter (pR-pFraB) Strength Measured by Luminescence

Effect of Fusion

Attention should be given to the y axis and the scale of the relative light units. The production in the fusion promoter (figure 3) is in the millions, whereas the other promoters characterized (figures 1 and 2) are in the hundreds of thousands, which is a tenfold difference. We first theorized that this could be due to the pR promoter overpowering the repression of pFraB, but the pR promoter by itself does not show such magnitude. We finally concluded that this overexpression could be due to the presence of two constitutive promoters stitched together, which could overpower fraR repression of pFraB.


Proteins

LAL Assay

Our plans deviated from original because of unexpected delays of a phage cloning kit due to the pandemic. Therefore, we were not able to test our system using phage to infect bacteria and then testing the product. Instead, we had to test our system in parts, one to determine promoter efficacy, which was our characterization, and one to determine the effects of the anti-lipid A proteins.

Due to time constraints, we only had the chance to test our constructs with one endotoxin detection kit- the LAL assay. We lysed our TOPO constructs and control, diluted, and compared to a control strain that had unmodified lipid A. We converted figure 4’s axis from quantitative absorbance data to Endotoxin Units, which were based on standards that the kit provided.

LAL Assay Graph
Figure 4. Endotoxin Units of Construct Lysates via LAL Assay

Almost all of our anti-lipid A proteins decreased the amount of lipid A compared to the control, with the exception of pFraB-LF. It should be kept in mind that LF (lactoferrin) and LptA are the only two proteins whose mechanism of action is to physically bind lipid A (binding proteins). Therefore, it makes sense that pC, whose characterization data showed great levels of expression, would increase the efficacy of irreversible binding proteins. The other proteins, whose mechanism is modification of lipid A, are much less consistently affected by the pC promoter.

The one that appears to work the best is pFraB-LpxR, however, the full pathway, LpxEFR does not seem to be much more effective. This holds true for PagL/PagP and PagLP which is another pathway divided into parts. This implies that the effects of these anti-lipid A binding proteins are not necessarily additive.