Results
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We intend to build a criticality detector and explore the potential application of it. The criticality de-tector, consisting of a light-sensing part and a negative feedback circuit, is designed to generate pulse output when the input exceeds the threshold. The binary system uses serine recombinases to record the number of pulse from the detector. The dosage control system is built to produce spe-cially designed antimicrobial peptide in a pulse manner for treating dental caries.
In the result page:
1.Photos of gel electrophoresis (confirming the successful construction of all gene circuit)
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2. CI binding site insertion with whole plasmid PCR
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3. Experiments proving the efficiency of the antimicrobial peptide
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Criticality Detector_Light-Sensing Part
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part: Promoter + Cph8
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 2612 bp
Construction succeeded!
part: RBS + ho1
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 1083 bp
Construction succeeded!
Results
part: RBS + pcyA
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 1107 bp
Construction succeeded!
part: RBS + ho1 + RBS + pcyA + terminator
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 2021 bp
Construction succeeded!
Results
part: RBS + ho1 + RBS + pcyA
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 1884 bp
Construction succeeded!
part: Promoter + RBS + Cph8 + RBS + ho1 + RBS + pcyA + terminator
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 4327 bp
Construction succeeded!
Results
part: Pompc+Cl binding site+RBS+GFP+terminator
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 1346 bp
Construction succeeded!
part: Pompc+Riboregulator+C1+B0010+Pompc+taRNA+B0010
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 1550 bp
Construction succeeded!
Results
part: Pompc+taRNA+B0010
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 598 bp
Construction succeeded!
Colony pcr
part: APP2 (48bp)
part: Bac8c (30)
part: CAP (28bp)
part: CAP-app2(82bp)
Results
part: CAP-Bac8c (64bp)
Colony pcr
CI Binding Site Insertion_A Brand-new PCR Strategy
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Inspired by the site-directed mutagenesis and the overlap PCR, we developed a two-step whole plasmid PCR strategy that enables precise insertion of a relatively long DNA segment in any site of interest. For the great convenience this strategy could bring, we decided to show it in our result. Advantages are listed below:
1.It saves a lot of time from molecular cloning. Teams of iGEM always spend a lot of time on the digestion with restriction endonuclease to obtain the target DNA and the ligation to construct a composite part. Also, adding a tag to a basic part using ordinary overlap PCR requires the subsequent reconstruction of all the composite parts containing that basic part. However, with the assistance of our PCR strategy, teams are able to get a whole plasmid ready to be transformed after the insertion at a predefined site.
2. It enables the insertion of a relatively long DNA segment. The site-directed mutagenesis could be used for insertion in a whole plasmid, but the insertion is merely several base pairs long. Our PCR strategy is proved to insert a DNA segment (i.e. the 46-bp long CI binding site) which is longer than what the site-directed mutagenesis could provide. Theoretically speaking, it could even afford insertion of longer DNA segments.
We successfully inserted the CI binding site into the specific site after failed many times with the ordinary assembly strategy.
Step1:The first pair of primers are adjacent to each other with opposite direction. After amplification by PCR, circular plasmids are “opened” at a specific cite of interest and linear plasmids are obtained (see Fig.1).
Fig.1 The first pair of primers "open" the circular plasmid at a specific cite.
Fig.1 The first pair of primers "open" the circular plasmid at a specific cite.
CI Binding Site Insertion_A Brand-new PCR Strategy
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This step could not be omitted according to our result. RFP with promoter on pSB1C3 was selected to test the necessity of this step. With the help of specially designed primers for step1 and step2, a 37-bp segment would be inserted into the middle of the promoter, which would prevent the colony from becoming red. E. coli transformed with plasmids produced by a two-step PCR was grown on the left plate; E. coli transformed with plasmids produced by an one-step PCR (without step1) was grown on the right plate (see Fig.2). Results showed that most insertion failed without the first step while most insertion succeeded with a two-step strategy, which demonstrated the significance of the first step.
Fig.3 The second pair of primers conduct insertion and back-splicing.
Fig.3 The second pair of primers conduct insertion and back-splicing.
