Difference between revisions of "Part:BBa K1642011"
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Based on Pdark and HR, we constructed an improved biodesalination system which depends only on the switch between white light and darkness. In the growth stage and working stage, we provide white light, while in the induced expression stage the light source is removed. Darkness leads to starvation and the starvation can inhibit active export of sodium, which is essential for biodesalination in the working stage.(described l in section transport module). The biodesalination process controlled by Pdark is shown in Figure 1.4.3.
 
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<center>{{ Template:SJTU-BioX-Shanghai/Figure
 
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| figure = [[File:SJTUB biode(3).png| 410px | frameless ]]
 
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| id = 1.4.3
 
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| label = Biodesalination process controlled by Pdark.
 
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| descr = In the growth Stage, cyanobacteria grow to a log-phase; in the induction stage, darkness induces the expression of halorhodopsin, and additionally pushes cyanobacteria to starvation status; in the working stage, engineered cyanobacteria absorb sodium chloride under natural light.
 
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Revision as of 01:31, 19 September 2015

Expression of Halorhodopsin under the control of Pdrak

Figure1. Control mechanism of Pdark. (a) Under natural light, the green light component induces phosphorylation of CcaR, and the binding of it to Pdark prevents the binding of RNA polymerase, thus inhibiting expression of halorhodopsin. (b) Under darkness, RNA Polymerase binds to Pdark as it does in E.coli and initiate the transcription of halorhodopsin.

Pdark-HR is a plasmid used to test whether the protein halorhodopsin under the control of P dark works as we expect.

Up and Down are sequences used for homologous recombination.

Pdark(BBa_K1026009) is a “dark-sensing” promoter combines PcpcG2 and a constitutive promoter from E.coli. Pdark contains binding site of CcaR in PcpcG2, which overlaps with the binding site of RNA polymerase. Therefore when phosphorylated CcaR binds to it, the binding of RNA polymerase to Pdark will be blocked and transcription can’t be initiated.

RBS is the RNA binding site, which will hopefully increase the expression level of HR.

Halorhodopsin (HR) proteins are light-driven inward-directed chloride pumps from halobacteria. We use this as our biodesalination driver which confers cyanobacteria the ability to absorb chloride to a significant degree.

Terminator is the transcription terminator for the E.coli RNA polymerase.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 611
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3215
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 481
    Illegal NgoMIV site found at 783
    Illegal NgoMIV site found at 942
    Illegal NgoMIV site found at 1263
    Illegal AgeI site found at 1134
  • 1000
    COMPATIBLE WITH RFC[1000]