Team:Edinburgh/Cell Display

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 proteins of interest to carrier proteins which are naturally found on the outer membrane.

Outline

Berkeley 2009 tried several different carrier proteins with several different passenger enzymes, and had success in many areas. However, when they tried attaching cellulases, they weren't so 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).

We will try a different carrier. <partinfo>BBa_K265008</partinfo> by UC Davis 2009 is a synthetic, codon-optimised sequence, based on [http://www.ncbi.nlm.nih.gov/nuccore/AF013159 GenBank AF013159] and coding for the first 211 and last 97 amino acids of ice nucleation protein (INP, normally coded by the 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-based cell display. After [http://www.sciencedirect.com/science/article/pii/S016777991000199X Van Bloois et al (2011)]

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):

• Use the entire INP protein; fuse at the C terminal
• Delete the INP internal domains; fuse at the C terminal
• Delete all of INP 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.

Example 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>

Genetic instability

In order to display several different proteins on one bacterium, it will be necessary to have several copies of the INP gene fused to different enzymes. The presence of repeated sequences on a plasmid can lead to genetic instability.

This will not be a problem in the JM109 lab strain, which lacks an important recombination enzyme. 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

Flagellar display could conceivably be used to focus our enzymes into a more concentrated space, perhaps increasing synergy and efficiency. However, it is a trickier technology...

The fusion is done as an insertion into the fliC protein. This may be problematic in practice. I'm not sure this technology has been used for full proteins. [http://peds.oxfordjournals.org/content/10/11/1319.full.pdf+html Westerlund-Wikstrom et al (1997)] managed a 302 peptide insertion.

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).

• Westerlund-Wikstrom B, Tanskanen J, Virkola R, Hacker J, Lindberg M, Skurnik M, and Korhonen TK (1997) [http://peds.oxfordjournals.org/content/10/11/1319.full.pdf+html Functional expression of adhesive peptides as fusions to Escherichia coli flagellin]. Protein Engineering 10(11): 1319-1326.