Team:OhioState/Proof Of Concept
Proof of Concept
Phage Cloning and Lysate Plan
Initially we planned on cloning our constructs into lambda phage by using the Lambda ZAPP II kit from agilent. However due to COVID-19 delays we were unable to receive the kit in time to clone in lambda phage in a timely fashion. We then attempted to clone into phage without the kit. This was unsuccessful. In order to test whether our constructs would affect the immunogenicity of the cells we TOPO cloned our 16 constructs into E.coli K-12 Strains and then created a cell lysate by exposing 2 mL of a washed overnight cultured to 4 ml of a 1 M Tris-HCl, 1% EDTA, and lysozyme buffer at a pH of 8, and then sonication for 1 minute at the highest settings. This lysate was diluted appropriately and used for endotoxin detection assay.
Limulus Amebocyte Lysate Assay
The LAL assay is used to detect the presence of LPS. It is derived from the hemolymph of horseshoe crabs, which contain cells known as amebocytes which cause the clotting and congealing of hemolymph in response to endotoxin. The assay is sensitive to the pico-micro gram scale of endotoxin detection. The amebocytes cells are not live cells but lysed and the lysate is used. Factor C, Factor B, and proclotting enzymes are all zymogens, which are inactive until cleaved. When the LPS is introduced and binds to Factor C, Factor C will autoclave and become active to cleave Factor B. The rest of the pathway follows this process and produces a polymer gel-like substance. The Pierce LAL Chromogenic Endotoxin Quantitation Kit was used, which gives quantitative data using a color-changing substrate.
Results and Discussion
Both promoter systems were tested, pFraB and pR-pFraB (pC) along with a control strain that had no anti-lipid A proteins. We saw a significant decrease in endotoxin binding across almost all of our anti-lipid A proteins, save for pFraB-LF, compared to the control strain. This indicates that most of them reduced the endotoxin’s binding ability somewhat.
The results were also consistent with the mechanism of our proteins. Both LptA and LF bind proteins, while the other 6 modify chains on the structure of lipid A. When comparing the high transcription pC promoter to the pFraB promoter between the binding proteins, we saw there was a noticeable decrease whereas it was much less consistent across the modifying proteins. This implies that when we increase expression, our binding proteins, the ones that would be bound to the lipid A, would be more effective as overall concentration increases. This shows that not only do we have expected results, but also results that match the mechanism of our proteins.
While we cannot fully equate this to immunogenic response in vivo, we can extrapolate this to imply that this would be successful in reducing immunogenic response by hindering binding to TLR4 receptors, due to decreased amounts of endotoxic activity present.
HEK Blue Cell Detection Assay Overview (Future Plans)
The HEK Blue cell assay helped determine the effectiveness phage of our outcome by examining the amount of reduction of immunogenicity of LPS. In detail, we will apply HEK blue cell assay to detect the presence of modified/bound LPS based on TLR4 activation. When TLR4 was triggered by LPS, inflammatory responses happen and induce SEAP reporter genes. The Quantin Blue medium was added in the media and produced a blue and purple color that can then identify NF-kB inflammatory responses. The quantitative analysis of endotoxin concentration was measured by the absorbance at 620-655 nm. We can use phage lysates from each of our phage constructs as samples. Negative controls would be unmodified phage lysate and uninfected cell lysate. This would allow us to see how the human TLR4 receptor would react to our phage infecting and killing the bacteria and confirm the results of our LAL Assay.
Sources
(1) - Sharma, Jill, et al. "Reporter Cell Assessment of TLR4-Induced NF-κB Responses to Cell-Free Hemoglobin and the Influence of Biliverdin." Biomedicines 7.2 (2019): 41.
(2) - Lenski, Richard E. "Dynamics of interactions between bacteria and virulent bacteriophage." Advances in microbial ecology (1988): 1-44.
(3) https://www.thermofisher.com/order/catalog/product/88282
