Part:BBa_K2389060

gvpA+C under lac promoter

gvp3.0 is an engineered operon consisting of different alleles of gas vesicle protein (gvp) genes, gvpA and gvpC. Both genes encode for the main structural proteins of gas vesicles, though, the different versions of gvpC (gvpC16 and gvpC20Ψ) allow for the production of varied gas vesicle phenotypes. Expression of the gvp3.0 construct is under control of the lac promoter (pLac), allowing inducible expression using IPTG in the presence of cAMP.

Gas vesicles are impermeable to water and entrap ambient gases within their structure. This property leads to an overall change in cytosolic density which has been seen to confer buoyancy to microorganisms which synthesize gas vesicle proteins. Our gvp3.0 construct is based off of the wild-type cluster found in the cyanobacterium Planktothrix rubescens. We have maintained this wild-type sequence of the gvp gene repeats, as this arrangement has been found to be conserved in the buoyant strains of P. rubescens. We hypothesize that the preservation of this sequence may have influence stoichiometric control of gvp subunits, or transcriptional efficiency.

As of now, the gvp3.0 construct is still undergoing thorough characterization. Though, the BioBrick has been sequenced verified and submitted to the iGEM BioBrick Registry as part of Team UAlberta’s Parts Submission. Currently, we have performed a series of buoyancy assays with gvp3.0 transformed E. coli. Though, we have been getting inconclusive results as different instances of our buoyancy assays have shown different indications of buoyancy, while some have not. A promising sign which may indicate that the gvp3.0 construct is successful in generating gas vesicles when expressed are our transmission electron microscopy (TEM) micrographs. With the help of Dr. Xuejun Sun and Mrs. Priscilla Gao, we have obtained images of our gvp3.0 transformants where structures, that resemble those of gas vesicles (Wang et al.) are observed. However, it will be necessary to repeat imaging of our samples with TEM with a separate biological replicate to confirm that gas vesicles are being synthesized.

References
Wang, Tianhe, et al. “Floating Escherichia coli by expressing cyanobacterial gas vesicle genes.” Journal of Ocean University of China, vol. 14, no. 1, Dec. 2014, pp. 84–88., doi:10.1007/s11802-015-2344-3. 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 AgeI site found at 200
  
• 1000
  COMPATIBLE WITH RFC[1000]