Coding

Part:BBa_K1499000

Designed by: Jotthe Kannappan   Group: iGEM14_StanfordBrownSpelman   (2014-08-27)

wssF gene from wss operon

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

Usage and Biology

wssF is one of the four genes isolated from Pseudomonas fluorescens responsible for acetylating cellulose (the others being wssG-I, K149001-3). 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. wssF has been purported to present acetyl groups to wssGHI [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 104
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

Verification

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

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

The part was sequence verified after insertion into pSB1C3 and before submission to the registry with two reads using VF2 and VR (Figure 2).

Figure 2. Sequencing alignment showing the proper insertion of wssF into pSB1C3

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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