Part:BBa_K2555004
PcopA-RiboJ-RBS-sfGFP-PBAD-RBS-CueR
BBa_K2555004 contains 2555000 and 25550002.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.BBa_K2555002 contains pBAD (L-arabinose inducing promoter) with RBS and CueR (a transcription factor that binds onto pCopA of BBa_K2555000). E. coli cells use CueR to regulate the cytoplasmic copper concentration. Arabinose concentration controls the expression of CueR in the construct.CueR behaves as a net activator or a net repressor under different copper concentrations(4)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2447
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2386
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2221
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1121
Illegal SapI site found at 2203
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
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The results of dynamic range of BBa_K2555004 with different arabinose concentrations after five hours. Error bars show standard deviation of three repeats
The figure showed that under o.1 mM arabinose BBa_K2555004 has the biggest dynamic range, so the amount of CueR under o.1mM arabinose may has positive effect on activation of pCop A.
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The results of a plate reader experiment with different copper and arobinose concentrations after five hours. Error bars show standard deviation of three repeats
We linked BBa_K2555000 (optimized BBa_K1555000) with BBa_K2555002 to construct BBa_K2555004. The relative fluorescence intensity, the increasing rate of relative fluorescence intensity and dynamic range of BBa_K2555004 under 0.1 mM Arabinose were all significantly 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.
(4)Danya J. Martell, Chandra P. Joshi, Ahmed Gaballa, Ace George Santiago, Tai-Yen Chen, Won Jung, John D. Helmann, and Peng Chen. Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription.Proc Natl Acad Sci U S A. 2015; Nov 3; 112(44): 13467–13472.
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