Team:Edinburgh/Cell Display

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Revision as of 10:06, 1 July 2011

An obvious type of bioreactor is an E. coli cell that displays the desired proteins on its outer membrane. This type of display is called cell surface display.

This works by fusing the protein of interest to a carrier protein which is naturally found on the outer membrane.

Notes for standard display

Berkeley 2009 Parts may or may not be helpful: they tried several different carrier proteins. When they tried attaching cellulases, they weren't too successful - of the two quantified cellulases, one worked just as well without the carrier (Cel5b) and the other didn't work (Cel9a, as compared to negative control).

<partinfo>BBa_K265008</partinfo> is the first 211 and last 97 amino acids of ice nucleation protein (INP, coded by [http://www.uniprot.org/uniprot/O30611 inaK] gene) from [http://en.wikipedia.org/wiki/Pseudomonas_syringae Pseudomonas syringae]. It seems promising as a carrier of enzymes. [http://www.sciencedirect.com/science/article/pii/S016777991000199X Van Bloois et al (2011)] speak highly of INP. Fusions are carried out at the INP C terminal.

Three strategies for INP cell display. After [http://www.sciencedirect.com/science/article/pii/S016777991000199X Van Bloois et al (2011)]

We could be the first team to attempt Ice Nucleation Protein as a carrier. INP has major domains at its N and C terminals, as well as a number of internal repeating domains. There seem to be three strategies for using INP (see figure):

• Delete nothing; fuse at the C terminal
• Delete the internal domains; fuse at the C terminal
• Delete everything except the N domain; fuse at the new C terminal

<partinfo>BBa_K265008</partinfo> should be suitable for the 2nd strategy.

Linkers

It may be desirable to create linkers between the carrier and the protein of interest. BioSandwich could be ideal for this.

Complete system

A complete 3 cellulase system would contain:

• Promoter -- RBS -- <partinfo>BBa_K265008</partinfo> -- Linker? -- <partinfo>BBa_K392006</partinfo>
• Promoter -- RBS -- <partinfo>BBa_K265008</partinfo> -- Linker? -- <partinfo>BBa_K392007</partinfo>
• Promoter -- RBS -- <partinfo>BBa_K265008</partinfo> -- Linker? -- <partinfo>BBa_K392008</partinfo>

Notes on genetic instability

The comments on the Phage Reactor page also apply here: there is a potential for genetic instability due to the presence of repeated sections of DNA in the system. However, this will not be a problem for our JM109 lab strain, which lacks an important recombinase. As for the use of this technology in industry, it will be possible to overcome this problem simply by synthesising coding sequences with as many altered (but synonymous) codons as possible.

Notes for flagellar display

This may be tricky...

References

• Van Bloois E, Winter RT, Kolmar H, Fraaije MW (2011) [http://www.sciencedirect.com/science/article/pii/S016777991000199X Decorating microbes: surface display of proteins on Escherichia coli]. Trends in Biotechnology 29(2): 79-86 (doi: 10.1016/j.tibtech.2010.11.003).

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