Fimbrial Mediated Attachment

The fimbrial cap protein fimH is the main anchoring element for our experimental design. Due to it's affinity to bind to surfaces, FimH has been frequently utilized in synthetic biology research as a tool to help localize bacterial species to surfaces such as mannose and nickel beads. Our project aims to modify the binding capabilities of FimH to allow it to anchor engineered E.coli cells to colorectal tumors. This is achieved through the use of protein tags, specifically the histidine and Synaptosomal- Associated Protein/histidine combination tags, which are intended to increase the binding affinity of the fimbriae to foreign surfaces such as a tumor microenvironment. The tags assist binding by allowing the FimH protein to effectively various target signals naturally expressed on the surface of tumors without conformational or binding strain.

Transformation and Sequencing of Engineered Tags


In order to study fimbrial attachment mediated by protein tags, the team had to first successfully implement the tags in the E.coli strain BL21. Following plasmid design, transformations of the tags into E.coli cells were performed by the Genescript Biotech Corporation. To ensure that the correct plasmid DNA had been incorporated into the bacterial system, Sanger sequencing was performed on the bacteria.

pRSF_SNAP_His_FimH (SNAP + His Tag)   pRSF_His_FimH (His tag)

Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis


Following transformations of the pRSF_SNAP_His_FimH and pRSF_His_FimH plasmids into BL21 E.coli cells, cultures grown from the transformed bacteria were diluted to separate cultures with dilutions of 1:50 and 1:100. The cultures were then induced with Isopropyl β- d-1-thiogalactopyranoside (IPTG), an allolactose mimic, which is able to activate the expression of the FimH protein containing the Histidine and SNAP-Histidine tags. After induction, a sodium dodecyl sulphate–polyacrylamide gel electrophoresis, or SDS page gel was performed on the proteins. SDS page gels are separation techniques which use electrical currents to separate proteins based on their lengths. The basis of the separation is the higher mobility of smaller, shorter proteins compared to larger, bulkier proteins. In our case, we utilized separation based on protein length as a means of verification that the SNAP-Histidine and Histidine tags were properly expressed on the BL21's FimH. This was done by drawing comparisons between the measured lengths of proteins extracted from the transformed bacteria to the known lengths of the protein tags.

Nickel Bead assay


Following transformations of the pRSF_SNAP_His_FimH and pRSF_His_FimH plasmids into BL21 E.coli cells, a nickel bead assay was run on the cells. In the assay, bacterial cultures of the transformed cells were diluted 1:200 and induced with Isopropyl β- d-1-thiogalactopyranoside (IPTG) to activate expression of the modified FimH protein. Following induction, the cells were introduced to Ni (2+) NTA beads and then visualized under a microscope. A culture of untransformed BL21 was also run through the protocol alongside the modified BL21 as a negative control. The purpose of this assay was to compare fimbrial mediated attachment in E.coli cells containing native and modified fimbriae. Comparisions were drawn between the unmodified BL21 cells, BL21 cells containing histidine tagged fimbriae, and BL21 cells expressing fimbriae containing both a SNAP and a histidine tag. Binding was visualized using a standard laboratory microscope. Future imaging efforts may entail the use of electron microscopy and advanced visualization methods to view binding.

Localization of the modified fimbriae around the nickel beads should resemble the following.

Note: the above figure is not to scale and is depicted disproportionately to demonstrate the binding of modified fimbriae to the nickel bead surface.

BL21 cells containing native fimbriae

BL21 cells expressing fimbriae containing a Histidine tag

BL21 cells expressing fimbriae containing both SNAP and Histidine tags

Conclusions and Further Steps:


Sequencing results from BL21 cells indicated that the pRSF_SNAP_His_FimH and pRSF_His_FimH were successfully taken up by BL21 cells during transformation. This indicated that the plasmid design was compatible with the cells. However, results obtained from the Nickel Bead assay and the SDS page gel indicate that the plasmid constructs need to be modified in the future to optimize binding and expression.

In the SDS page gel, the lengths of the protein are not clear from imaging taken of the gel. This error may be due to procedural errors such as inefficient IPTG induction conditions which may have reduced the expression of the fimH protein in the bacteria. However, another significant contributor may be inefficiences in the construct design which contribute towards inconsistent expression of the modified FimH protein. Going forward, we hope to optimize this protocol such that separation of proteins is efficient leading to clear visualization of beads such that the lengths of modified protein can be definitively determined.

In the Nickel Bead assay, the clear and black rods represent bacteria. Minimal binding of BL21 cells transformed with histidine tag and histidine-SNAP tag combination plasmids was observed during the assay. This is because on the microscope images, a low amount of rods can be seen clustering around the nickel beads. Rather, the majority of bacterial density appears to reside in the empty field outside of the bead area. While the number of rods may be affected by the design of the experimental protocol and the instrumentation used for visualization, the most likely explanation behind the low binding affinity is the design of the plasmids encoding for the protein tags. However, as the plasmid design is not finalized and can be altered, our team looks forward to optimizing protein tag function through continued iterations of the assay. For example by optimizing the buffer used in the experiment, any screening effects on the binding of bacteria to the Ni(2+)- NTA beads could be reduced. Other optimizations include varying the beads used in the experiments, such as the use of magnetic beads to allow for bound bacteria to be seperated from the solution and for binding to be quantified.

Hypoxic Activation of OxyR and Reporter Release

Hydrogen Peroxide Induction


The purpose of Hydrogen Peroxide Induction were two-fold. First, we were looking for the maximum concentration of hydrogen peroxide that would not inhibit the growth of our transformed bacteria. Secondly, we wanted to activate the OxyR protein in order to control the expression of the β-galactosidase enzyme.

It was found that different concentrations affected each OxyR controlled gene differently. To serve as a baseline, we were looking for the concentration at which the cells would achieve an OD of around 0.6. The most resistant gene was dps which was able to reach the OD in a max concentration of 1 micro molar. There was a tie between the KatG and grxA as the second most resistance transformed bacteria. KatG was the only gene to show a significant difference with increasing concentration.

Figure 1. OD600 vs. time plot for katG

The exponential trendline gives us the doubling time for the culture at these concentrations.

Figure 2. katG doubling times at H2O2 concentrations

The dps, grxA, and control plots are shown below.

Figure 3. OD600 vs. time plot for dps

Figure 4. OD600 vs. time plot for grxA

Figure 5. OD600 vs. time plot for pUC-19 control

β- Galactoside Assay


The purpose of the β-galactosidase assay was to see if the β-galactosidase enzyme was produced and able to turn bacterial cells blue when in supplied with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (Xgal).

From left to right, the following plates were prepared to probe OxyR activity: 1 mM of H202 alongside DH5-α transformed with the grxA gene insert, 500 uM of H202 alongside DH5-α transformed with the grxA gene insert, 100 uM of H202 alongside DH5-α transformed with the grxA gene insert, 100 uM of H202 alongside DH5-α transformed with the dps gene insert, 100 uM of H202 alongside DH5-α transformed with the KatG gene insert. katG produced the most significant data.

The results of the β- Galactoside Assay were inconclusive as a color change was not able to be enacted in all transformed DH5-α cells (this includes those containing our designed OxyR gene inserts and those containing a puc19 control plasmid

Conclusions and Further Steps:


To improve upon our H2O2 induction system, we plan to optimize the sequence for the OxyR binding site. We generated our original OxyR binding site plasmids by inserting the upstream sequences of genes controlled by OxyR (ex. grxA, dps, and katG). We aim to narrow down the sequence for the binding site to improve the affinity between OxyR and the specific sequence. Based on our modeling results, increasing the binding affinity would improve our system’s ability to detect changes in H2O2 concentrations. Furthermore, we want to use OxyR to control the expression of reporter genes other than lacZ-alpha. For instance, it may be possible to have OxyR induce the expression of proteins that are more easily detectable than beta-galactosidase.