Team:Pittsburgh/Design

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Design

 

Introduction

Our initial goal for the project was to design a system that, when cancer cells are detected, produced a β-galactosidase reporter that would cause a color change in the stool. We initially explored two methods of detection: FimH/OxyR mediated response and quorum sensing mediated response. FimH is an E. coli fimbriae capping protein. OxyR is a transcription factor that up-regulates lacZα production through FimH binding and increase H₂O₂. Team discussions, meetings with our mentors and microbiology experts, and examining the current literature led us to pursue this path for our project.

Overall Process

1

Plasmid Design and Construction

To conduct desired experiments, we have designed gene reporter assays using the Benchling website to track the following OxyR regulated genes in the pUC-19 vector backbone: dps, grxA, and katG. Each OxyR regulated promoter is then designed to regulate the expression lacZα. Designs were subsequently submitted to Genescript for DNA synthesis. Plasmids were confirmed by DNA sequencing and investigative plasmid digestion assays.

The Native FimH is stimulated by its interaction with mannose. Past studies have shown that chimera’s of FimH with a hexahistidine tag could be stimulated by binding to Ni-NTA beads. The SNAP tag can be attached to a protein of interest and functions to tag the targeted antibodies. FimH his and SNAP tags were designed in benchling for our experimental use. Upon arrival, both fimH plasmids (histidine and SNAP tags) were transformed into competent cells and incubated in order to ready them for future experimentation.

2

FimH Binding

One of our plans to accomplish our goal is to express the pUC19 plasmids containing OxyR binding sites with plasmids which express our FimH-SNAP fusion protein. In order to express two plasmids, the two plasmids need to have different origins of replication and antibiotic resistance. As a result, we picked the pRSF-Duet backbone and cloned in the gene for the fusion protein. pRSF-Duet has a different origin compared to pUC19 and has kanamycin resistance instead of ampicillin. We attached the SNAP tag to the N-terminus of the FimH protein¹. We also created a His tagged version as a control. The SNAP tag codons were optimized for expression in E. coli.

After cloning in the SNAP_His_FimH and His_FimH fragments separately into the pRSF-Duet vector, we verified the resulting plasmid. Automated Sanger sequencing was performed. The figures show a portion of the sequencing result and where they match up to on the plasmid. Both of the figures show that the cloning process was successful since the sequencing matched up to our original plasmid sequence.

Next, the expression of the fusion protein was confirmed via a SDS-PAGE gel. The BL21 cells containing our plasmids were induced with IPTG for 3 - 4 hours. The gel is then stained with Coomassie. Our SNAP_His_FimH fusion protein should be around 53 kDA and our His_FimH protein should be around 35 kDA. Neither of the bands showed up in the induced lanes. This is likely due to the fact that we induced the cells at OD600 ≈ 0.9, which is too high. We grew the cells for 3 hours after inoculating an overnight culture and then induced it with IPTG.

3

OxyR Induction

OxyR’s transcriptional activity is directly regulated by H₂O₂. The cancer cell environment is hypoxic, which upregulates lacZα transcription via OxyR. In order to simulate hypoxic OxyR induction, we modeled experiments based on Rodríguez-Rojas et. al. and Schaefer et al. to determine the concentration of H₂O₂ required to produce enough lacZα for a visible color change. The literature suggests that the minimal inhibitory concentration for H₂O₂ is 1 mM². This concentration was subsequently used as a reference for 30-minute killing curves that show a clear dose-effect in survival rate. We tested this by exposing cultures of the three OxyR regulated genes to H₂O₂ concentrations ranging from 50 μM to 1mM and measuring OD₆₀₀ every thirty minutes for three hours. Cells were then pelleted and exposed to a solution of lysis buffer and x-gal; OD₄₂₀ was measured to determine magnitude of color change³.

References

1. Cole, N. B., Site-Specific Protein Labeling with SNAP-Tags. Current Protocols in Protein Science 2018,70 (1), 1-30.
2. Rodríguez-Rojas, A. K., Joshua Jay; Johnston, Paul R.; Makarova,Olga; Eravci, Murat; Weise, Christoph; Hengge, Regine; Rolff, Jennss, Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress. PLOS Genetics 2020, 3.
3. Schaefer, J. J., Goran; Kotta-Loizou, Ioly; Buck, Martin, Single-step method for β-galactosidase assays in Escherichia coli using a 96-well microplate reader.