Part:BBa_K228003:Experience

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2011 iGEM Team Peking_R

BBa_K228003 is modified by 2011 iGEM Peking_R team as a demonstration of RNA toolkit functions and RBS calculation developed by our group.

When the bistable switch part is transformed into DH5α strain, green colonies and red colonies were 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.

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.

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.

Thus, a library mutating the RBS of cI434 gene in BBa_K228003 part is constructed via site-directed mutagenesis method to verify the conformity of our model with experimental results. (Figure 2) Each plasmid of the library is transformed into E. coli DH5α strain separately harboring the bistable switch logic device. After growing on agar plates, G/R ratio is calculated for each of the sequences in the library, several images of which are captured by the fluorescence stereomicroscope. (Figure 3) The ability of translation strength to regulate the switch’s behavior is thus verified.

Figure 2 Construction of bistable switch library via site-directed mutagenesis. Forward and reverse primers binding sites are schematized.

Figure 3 Experimental results of varying translation rate by altering the RBS sequence upstream of cI434 gene. Images were acquired by applying excitation light with wavelengths of 470nm (for eGFP) and 580nm (for mRFP) to the agar plate respectively and merging two emission images together. (A)Synthetic RBS with △G of about -3.029kJ/mol. Calculated proportion of “green” colonies is 0.95. (B)Synthetic RBS with △G of about -2.08kJ/mol. Calculated proportion of “green” state is 0.35. (C)Synthetic RBS with △G of about 0.79kJ/mol. Calculated proportion of “green” state is 0.21. Apparently, translation strength indeed has a significant role in regulating the device’s behavior.

Additionally, TPP down-regulated hammerhead ribozyme 2.5 is introduced into BBa_K228003 regulating gene expression of cI434 as the demonstration for the function of RNA controller. (Figure 4)

Figure 4 Construction of bistable switch library via site-directed mutagenesis. Forward and reverse primers binding sites are schematized.

We set two experiment groups for this part (BBa_K598024): one without addition of TPP and another with TPP sufficient for full induction of the RNA controller’s functions (self-cleavage of ribozyme). The experimental results are shown in Figure 5. It can be seen that the group with excess TPP (down-regulated translation strength of cI434 gene) indeed displayed bistability. Because the two states can be viewed as one without induction and one with full induction respectively, they can be mapped to the two ends of the translation strength(△G)-ligand concentration curve (shown in Figure 5). From the figure it can be seen that the change in △G between the two statesis also about 4kcal/mol. Therefore, the results of RNA controller regulation both met the parameter requirements for a functional bistable device and verified the accuracy of the mathematical model.

For the full characterisation of the device, please refer to <p><b>For the full characterisation of the device, please refer to BBa_K598000 BBa_K598002

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