Part:BBa_R0051:Experience

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Experience by [http://2012.igem.org/Team:NTU-Taida 2012 iGEM NTU-Taida]

We tried to modify pCI, the well-known regulator from lambda phage. The original design of pCI in iGEM community is pR, which is composed of OR1 in -10 region, and OR2 in -35 regions. The transcriptional factor CI would preferentially bind to OR1 and then OR2 with cooperatively binding, via C terminal domain interaction. In the normal physiological condition, the binding of OR2 would further tighten the binding of RNA polymerase to pRM promoter and stimulate its own transcription. The binding site -10 upstream of starting point is critical to the function of the repressor. Under this notion, it seems more reasonable to pick a sequence unit with a higher affinity in -10 region. Thus we incorporate OL1, only one base pair different from OR1, and 10 times higher affinity, in our modified pCI repressor, with OR1 in -35 region.

Reference

1. Dodd, J.B. Shearwin; Egan, J.B.; Egan, JB (2005). "Revisited gene regulation in phage lambda". Curr Opin Genet Dev 15 (2): 145–152.
2. Direct demonstration and quantification of long-range DNA looping by the λ bacteriophage repressor, Nucleic Acids Res. 2009 May; 37(9): 2789–2795

User Reviews

UNIQ93b930c7b761c661-partinfo-00000001-QINU

••••• Antiquity

This review comes from the old result system and indicates that this part worked in some test.

• Tokyo_Tech

Transformation and o/n culture is Okay, but we couldn't extract plasmid by miniprep. We couldn't detect band by Electrophoresis. We don't know why, but this occured 2 times.

•••• Aberdeen_Scotland 2009

The initial gel worked although the obvious confirmation was not possible due to the very small fragment size (49bp).But later it was confirmed following its usage by forming the part BBa_K182002 through the PCR gel analysis and the sequencing.The plasmid miniprep also worked.

•• Seu_O_China 2012

BBa_R0051 does not work well due to its rather small size. We transformed it for three times, and none of them succeed. We designed primers to PCR it out from K091230 successfully, but failed to perform the digestion since it is too small to extract from gel. It may work better if adding some nonsense sequence before the promoter. UNIQ93b930c7b761c661-partinfo-00000006-QINU