Partnership

Meeting with XJTLU-CHINA

Getting acquainted

This year, we found that another iGEM team XJTLU is also working on expressing fusion protein. Specifically, they plan to engineer E. coli to express csgA protein at high levels and immobilize the bacteria into a filter. In that way, the heavy metal ions in the wastewater can be captured by csgA protein that is expressed by E. coli located in the filter. Overall, they plan to develop a biofilter core to replace current industry filter cores for heavy metal wastewater.
Therefore, we decided to arrange a meeting to share each other's ideas on this topic and discuss the way we might work together.

Discussion

After we both introduced our projects, we exchanged our thoughts about each other's projects.

Questions & Advice From XJTLU-CHINA

  1. They would like to know how we will interact with and educate the public.

    A: We have made questionnaires to evaluate their knowledge related to antibiotics, probiotics, and microorganisms. We are also planning to make some handouts and deliver a lecture about it in a high school.

  2. They were interested in the specific design of our plasmids. Specifically, it's about how and why we choose each part of the plasmid for surface display.

    A: We applied three microorganisms and the whole team was divided into three groups, each responsible for E. coli, yeast cells and Lactococcus lactis. All groups applied a strategy that conjugates the targeted nanobody with a surface protein that is to be expressed, transported and located in the extracellular space. The surface protein and the targeted nanobody were connected with a linker sequence, and each nanobody is marked with at least one protein tag for their identification. We also provided links to iGEM parts so they can check them directly.

Questions & Advice From DKU

  1. Will the biofilter be blocked due to E. coli proliferation?
    A: The microorganisms are immobilized in filters, and the filtration condition may not allow E. coli to grow at such a speed.

  2. The csgA protein's ability to absorb heavy metal ions under different conditions (like pH) can be tested through experiments. For example, we can put csg protein into a certain amount of wastewater, then adjust the pH and check the remaining concentration of metal ions in time, so we can acquire a proper pH for the protein to capture heavy metal ions.

Cooperation plan

After the Q&A session, we discussed several key topics that might be helpful to the project design.

  • Model for bacteria growth
    XJTLU-CHINA has developed a model for predicting bacteria growth, and it might also be applied to our situation by adjusting some of the parameters.
  • Quantified expression of csg through controlling IPTG concentration
    XJTLU-CHINA intended to control the expression of csg protein by adjusting the amount of IPTG inducer, which can also be a helpful approach for our team to check the relationship between the cell-cell adhesion affinity and the number of expressed nanobodies. However, as we also applied the TlpA promotor, it may influence the actual expression to a great extent and cause a big difference from the theoretical calculation. As a result, this approach remains to be a concern.
  • Detection and measurement of displayed csg protein
    XJTLU-CHINA controlled the expression level by adjusting inducer concentration and estimated the expression by calculating theoretically, which might be a possible direction for us to consider.
  • The condition of csg and heavy metal ions after the death of E. coli cells
    We consider that the heavy metal ions will remain captured, as protein itself has a great affinity to the heavy metal ions. But, it remains to be seen whether the released protein will leak out from the filter, and this requires more work.
  • Supplement of experimental materials
    We would like to give special thanks to team XJTLU for providing us the csg sequence for wet lab.