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

UNIQ9b815886e0b2c655-partinfo-00000001-QINU

UNIQ9b815886e0b2c655-partinfo-00000002-QINU

Shenzhen_SZMS 2016

We met the same problem Tokyo Tech has faced back in 2007 that bacteria could grow on solid/liquid medium with Amp, but nothing was found in gel after miniprep and electrophoresis. This whole procedure was repeated at least 5 times. Meanwhile, some new bacteria did grow on the Amp medium after a couple of days R0051 plasmid was transformed. We would say that for some reason R0051 plasmid gave the host bacteria resistance to Amp and then disappeared. Pretty strange and annoying.

UNIQ9b815886e0b2c655-partinfo-00000004-QINU

BBa_R0051 3 Not understood WHU-China 2012

In our experiment, we used an intermediate protein cI and constructed an indirect pathway. Our device will respond to glucose, being activated at high concentration of glucose.In this way, we changed the function of the promoter R0051. The indirect regulatory pathway As you can see, in this pathway, protein cI will be expressed when CRP is activated at low glucose concentration.The large amount of cI repressed the promoter R0051 heavily. On the contrary, when glucose concentration is high, the promoter is derepressed. In this figure, the fluorescence of RFP generator promoted by PcstA decreased when concentration of glucose rose, meanwhile, the fluorescence of the indirect regulatory device increased with the glucose concentration, indicating that the device works as expected.In another word,the device we designed has a new function, activated by high concentration of glucose.

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UNIQ9b815886e0b2c655-partinfo-00000007-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. UNIQ9b815886e0b2c655-partinfo-0000000C-QINU