Part:BBa_K228003:Experience
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Applications of BBa_K228003
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.
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.
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)
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) displayed bistability. Therefore, the RNA controller(TPP ribozyme) is indeed capable of regulating the device’s behavior.
For the full characterisation of the device, please refer to BBa_K598000 BBa_K598002
Applications of BBa_K228003
2011 iGEM Team USTC-CHINA
Models
Figure 1: Constructions to modulate the ratio of toggle switches. The above bar represents pSB3T5, a low to medium copy BioBrick standard vector, where LuxpR (BBa_R0062) controls the expression of cI repressor which would influence the ratio of toggle switch. (Images by USTC-IGEM with TinkerCell)
Results
Verification of the original Toggle-switch from PKU
Figure3.A conlony of the bacteria with the original Toggle Switch exhibit two different states(4X Objective)
By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 8:25. It means the original Toggle Switch is not fit for our purpose, so we modify the original by luxPR-cI device.
Verification of the modified Toggle-switch
Figure4. The conlonies of the bacteria with the modified Toggle Switch exhibit two different states or just one state(4X Objective)
By calculating with the help of the fluorescence microscope, the ratio between the numbers of colonies with RFP and colonies with GFP ≈ 6:1. It is useful to our project design.
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