Coding

Part:BBa_K1499002

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-08-27)

wssH gene in wss operon

This part encodes wssH, a gene found in the wss operon of Pseudomonas fluorescens theorized to be involved in the acetylation of cellulose.

Usage and Biology

wssH is one of the four genes isolated from Pseudomonas fluorescens responsible for acetylating cellulose (the others being wssFGI, K149000, 1, 3). The wss operon in P. fluorescens contains 10 genes responsible for biofilm production, allowing the bacterium to colonize the air-liquid interface. The operon includes genes necessary for the biosynthesis of cellulose (including many with homologies to E. coli or G. hansenii, including minD, subunits of cellulose synthase, and a cellulose), and four (wssF-I) have been implicated as responsible for the acetylation of the cellulose polymer. wssH has been purported to form an acetylation complex associated with cellulose synthase [1].

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 366
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Verification

We successful isolated the four acetylation genes from P. fluorescens total DNA (Figure 1).

Figure 1. Pure genomic DNA was isolated from P. fluorescens and the four acetylation genes were amplified

However, this part sequence contained two BioBrick restriction sites, so we decided to have it synthesized in the backbone. DNA2.0 carried out this synthesis, and they verify the sequence before shipping.

Results

This part has been successfully cloned into the BioBrick backbone, but for expression in G. hansenii, our model cellulose-producing organism, we needed to use the multi-host shuttle vector pUCD4 (Figure 3).

Figure 3. Multi-host shuttle vector pUCD4

This procedure was more time-consuming than expected, as the pUCD4 plasmid is >10kb, the maximum size able to be purified by spin columns. Further work is needed to prove the activity of the wss genes in acetylating cellulose.

References

[1] Spiers AJ et al. (2013) Cellulose Expression in Pseudomonas fluorescens SBW25 and Other Environmental Pseudomonads in Cellulose - Medical, Pharmaceutical, and Electronic Applications. DOI: 10.5772/53736

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