Part:BBa_K2555000

PcopA-RBS-RiboJ-sfGFP

BBa_K2555000 including the promoter of cop A, ribosome binding site, self-cleaving RNA ribozyme RiboJ, and protein coding region-sfGFP. In E.coli. As a ATPase, cop A is regulated by a copper-responsive protein CueR(1). The promoter of cop A is more sensitive to copper than the other two copper-responsive promoter.RiboJ is an insulator commonly used in genetic circuits to prevent unexpected interactions between neighboring parts. Insulation with RiboJ may increase protein abundance. sfGFP increased resistance to denaturation, improved folding kinetics, and increased resistance to aggregation during refolding(2). sfGFP has been proven to be very useful for improved protein detection(3). Terminator L3SP22 is utilized for construction of a composite part.

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1121
  Illegal SapI.rc site found at 364
  

Method

In order to show that our synthetic bacteria have a fully functional part that works, expression measurements were made. The construct was linked to sf GFP. For the parts plate reader was done. A plate reader is able to take optical density (OD600nm) and fluorescent measurements over time. OD is a measure of bacterial growth over time and fluorescence is a measure of protein expression over time.

Results

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The results of a plate reader experiment with different copper concentrations after five hours. Error bars show standard deviation of three repeats

We optimized BBa_k1555000 to be BBa_K2555000. The relative fluorescence intensity of BBa_K2555000 under different copper ion concentrations was higher than that of BBa_k1555000, and the increasing rate of relative fluorescence intensity was higher than that of BBa_k1555000.

References:

(1)Outten FW, Outten CE, Hale J, O'Halloran TV. Transcriptional activation of an Escherichia coli copper efflux regulon by the chromosomal MerR homologue, cueR. Journal of Biological Chemistry 2000;275:31024-9.

(2)Andrews BT, Schoenfish AR, Roy M, Waldo G and Jennings PA. The rough energy landscape of superfolder GFP is linked to the chromophore. J Mol Biol 2007;373: 476-490.

(3) Cabantous S and Waldo G. In vivo and in vitro protein solubility assays using split GFP. Nat Methods 2006; 3: 845-854.