Part:BBa_K4212047

SDP4

L1

Introduction to this part

This part contains Hyperspank promoter, RBS3, GFPmut3B CDS, tL3S2P21 terminator.

Our Experiment Design

With 1A, 1B, and 1C successfully assembled and secured in glycerol stocks, the final step in allowing validation of our biocontrol measure lied in the construction of a reporter system to quantify the efficacy of the self-digesting circuitry. The approach adopted by Quijano et al. involves an anchor protein-GFP fusion to assess protein displaying abilities as well as plasmid retention following induction of sporulation. The authors used colony PCR combined with microscopy techniques to quantify effective protein display as well as plasmid retention post-germination.

Initially, we set out to follow the methodology outlined above, i.e. harnessing a fluorescent fusion protein to investigate the efficacy and impact of plasmid self digestion with our adapted circuitry. To that end, we designed primers for the amplification of GFPmut3b from STK053 in the STK toolkit, to be joined with the CotG linear fragment amplified as part of our Chitinase Display strategy in a Golden Gate reaction. Read more about our contribution to the STK for spore display systems here.

Figure 1. The scheme of our GFPmut3B assmebly design

Our Experiment Result

Within the course of the project, the L2A plasmid was successfully assembled as noted in figure xy which displays a cPCR result and restriction digest of positive colonies from the same. However, the L2B plasmid containing the GFPmut3B TU was not achieved, even after subsequent troubleshooting attempts. Due to time constraints, further progress was not made in assembling a self-digesting plasmid ready for expression in B. subtilis.

Figure 2. The PCR result of our gRNA assmebly design

References

[1] Franke, G.C., Dobinsky, S., Mack, D., Wang, C.-J., Sobottka, I., Christner, M., Knobloch, J.K.-M., Horstkotte, M.A., Aepfelbacher, M. & Rohde, H. (2007) Expression and functional characterization of gfpmut3.1 and its unstable variants in Staphylococcus epidermidis. Journal of Microbiological Methods. 71 (2), 123–132. doi:10.1016/j.mimet.2007.08.015.

[2] https://parts.igem.org/Part:BBa_K143015

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1743