Team:Lund/Engineering

iGEM Lund 2019

Engineering

On this page, we will showcase the engineering success behind our project

Figure 1. A flowchart showing the engineering cycle, with comments regarding how each step affected our project.


We were only available to achieve protein expression, but the following paragraphs. One of the reasons, apart from wanting to test combinations, for why we express multiple AMPs is because we expect to gain new knowledge along with the experiments. Many of them have never been previously expressed in bacteria, much less in E. coli. We need a system able to produce a gene product according to at least two quality parameters: a significant yield of soluble fusion protein and a usable yield of protease digested AMPs.


For producing the soluble protein, we try multiple fusion partners, Thioredoxin A (Trx A) and Glutathione S-Transferase (GST). Since many of the peptides are cysteine rich, we expect the disulphide bridge formation catalyzing Trx A to have the greatest solubility increase, but this hypothesis requires experimental validation. Given that we got protein expression with both of the fusion partners, we will have to test the solubility of both fusion partners before deciding on the most suitable one. We plan on doing this by sonicating the cells and then run SDS-PAGEs to see whether the protein is in the soluble or insoluble phase. There may also be other reasons for why a particular antimicrobial peptide is not expressed properly, and using a wider selection hedges us against unexpected issues.


When it comes to the AMP yield, meaning the yield from fusion partner digestion, we require experimental validation. The protease, enterokinase (EK), is a naturally occuring protease in mammals (humans, bovine). The region it recognizes and cleaves is hydrophilic, so it should be accessible for EK. However, the substrate data from Brenda indicates that while enterokinase is selective, it’s specificity varies with the chains, such that experimental validation is the most feasible approach within the scope of the project.


When we have produced cell extract containing AMPs, we will screen for which ones produce the strongest antimicrobial effect. Based on this, we will conduct a factorial experiment trying to determine if we can get a significant synergistic effect of combination therapy. We believe that with these objectives along with the proper research, design, building and testing, we will follow the engineering cycle in a way that will benefit the project.