Part:BBa_K3490021
Verify the function of EL222-pBlind and FLP-FRT systems.
According to research, HicA is a toxin promoting mRNA degradation, leading to the hibernation of bacteria, while HicB is an antitoxin that can neutralize the effect of HicA. Hence, through regulating the expression of hicA and hicB genes, we can control the growth of bacteria, hibernating the bacteria before the contact lens is used. Since contact lens are sealed during the process of production, it is impossible to regulate the growth switch through chemical induction; hence, we turned to external stimuli such as light, temperature, etc. At first, we used EL222 to regulate the system(a blue light-sensitive protein), pBlind (a promoter activated by EL222), FLP, and FRT to change the orientation of our promoter, which is activated by blue light. In other words, we have to illuminate the contact lens with blue light for 30 minutes before use. First, the production stage is when we are culturing our bacteria. Without any arabinose addition during culture, the bacteria will be able to grow normally. After the production process, we will put the bacteria and arabinose into the contact lens together, so the pBAD promoter will transcribe hicA, causing the bacteria to hibernate. When we need to resuscitate the bacteria, EL222 will be activated by blue light to induce pBlind promoter, and then FLP will be transcribed. FLP will act on the FRT sites to change the orientation of the constitutive promoter, as a result, the bacteria will start to transcribe hicB to neutralize HicA and thus continue to grow. We replaced hicA and hicB with GFP and OFP gene to build a test plasmid because it is much easier to observe the color change than to count the CFU. If the color of fluorescence changes from green to orange, the experiment is successful to prove that the design is feasible.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1573
Illegal SpeI site found at 1891 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1573
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 3035
Illegal NheI site found at 3058
Illegal SpeI site found at 1891 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1573
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1573
Illegal SpeI site found at 1891 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1573
Illegal SpeI site found at 1891
Illegal NgoMIV site found at 142
Illegal AgeI site found at 367 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2882
Illegal BsaI.rc site found at 3115
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