Difference between revisions of "Part:BBa K598002"

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Bistable Switch Mutant 68

Description

This part is one of the mutation libraries of bistable switch modifying the ribosome binding site (RBS) of cI434 gene.

The bisatble switch, which was inherited from the iGEM 2007 Peking Team, mainly consists of a positive feedback loop and a double-negative feedback loop. The expression of two mutually repressing repressors genes cI434 and cI are controlled by the promoters PR and PRM respectively. Promoter RM can be activated by CI and repressed by CI434. GFP and mRFP are placed downstream cI434 and cI as reporters of two states, respectively. In the state when CI is dominant, it can activate its own gene’s transcription and repress that of cI434, thus developing and stabilizing a stable high CI/low CI434 state, in which a red fluorescent protein (mRFP) gene co-transcripted with CI is expressed. Alternatively, when CI434 is dominant, a stable high CI434/low CI state will be established and GFP co-transcripted with CI434 is expressed to represent the this state. Each cell that bears the bistable switch is expected to express GFP or mRFP exclusively. The two states are believed to be stabilized over a long period while under certain circumstances one state may be turned over to the other.

When the bistable switch on pSB1C3 plasmid is transformed into DH5α strain, green colonies and red colonies could be observed. Interestingly, several mixed colonies could also be observed, which implied the random steady-state characteristic of the bistable switch (Figure 1). A ratiometric of the green colonies to the red colonies (G/R ratio) was calculated on the LB agar plate. We proposed that the G/R ratio is relevant to the translation strength of CI & CI434 genes, which means modulating the translation strength of one or more could result in different ratios of G/R under current architecture of bistable switch.

Figure 1 Images of colonies and individual cells bearing the genetic bistable switch. (A) A red colony and a mixed colony captured by fluorescence stereomicroscope. (B) Individual cell images in the mixed colony captured by laser confocal microscope. Each rod represents a single cell, expressing GFP or mRFP exclusively.

Modeling Confirmation

Library Construction

The bistable switch library modifying the expression of cI434 gene is constructed via site-directed mutagenesis method.(Figure 1)

Figure 1 Construction of bistable switch library via site-directed mutagenesis. Forward and reverse primers binding sites are schematized.

Primers are designed pairing the template (BBa_K228003) but replacing the RBS of cI434 gene with NNNNNNN. After PCR amplification, the PCR product was treated with DpnI and column-purified. The linear DNA was phosphoryated by T4 polynucleotide kinase and column-purified. The DNA was then self-ligated and transformed into DH5α cells. Thus, the library is established.

Experimental Data

Sequence and Features