Device

Part:BBa_K1913006

Designed by: Thomas Mathijs Swartjes   Group: iGEM16_Wageningen_UR   (2016-09-30)
Revision as of 11:32, 19 October 2016 by Tmswartjes (Talk | contribs) (→‎Usage and Biology)


434- and lambda cI balance operon + mRFP reporter

This part contains two viral genes: 434- and lambda cI (BBa_C0052 and part of BBa_K081007) expressed under the pBAD/AraC promoter (BBa_I0500). The 434 cI gene is not yet preceded by an RBS, allowing tuning of the cI protein balance. The 434 cI protein encoded in this part contains a C-terminal LVA tag causing more rapid protein degradation, the lambda cI gene in this part does not encode an LVA tag. Additionally this part contains an operon where mRFP (BBa_K081014) is expressed under a modified lambda cI promoter (BBa_I12006). This promoter is (according to the parts page) induced by lambda cI and repressed by 434 cI. This last operon also includes a LVA-tagged lambda cI (BBa_K081007) encoding gene, in theory establishing positive feedback of the modified lambda cI promoter. The two operons in this part were also submitted as separate parts:

  • 434- and lambda cI operon: BBa_K1913007
  • 434- and lambda cI balance RFP reporter: BBa_K1913016

The complete part is intended to respond with mRFP expression upon a change in strength of the inducible promoter (BBa_I0500). When - after a period of induction with L-Arabinose - the promoter is repressed with glucose, one expects an increase in mRFP (BBa_K081014) expression.


Usage and Biology

We inserted an RBS library (see design notes) upstream of the 434 cI gene and transformed it to DH5alpha. The transformants were expected to produce subpopulations in mRFP expression upon glucose addition. We tested this with flowcytometry (Figure 1).

T--Wageningen_UR--FACS_Thomas.jpg

Figure 1. Flow cytometry results for clone B6 and the constitutive mRFP expression control. The images on the left are histograms that show counts of bacteria with specific values of forward light scatter (FSC-H). The histograms on the right show counts of cells with fluorescence corresponding to mRFP. The mRFP fluorescence was measured with an excitation at 561nm, measuring emission at 620nm with 10nm bandwidth.

Although flow cytometry shows that two clones do not form subpopulations, this is no reason to assume that none of the clones are able to do so. We chose to include an 18-mer RBS library on purpose. We expect that if the system described here can lead to subpopulations, this ability relies on the RBS strength. As we have not been able to measure all RBS inserts, we cannot accept nor reject the possibility that the system described here works as intended.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1898
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 4249
    Illegal AgeI site found at 4361
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


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