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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
click here
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!
Criticality Detector_Light-Sensing Part
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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!
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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!
Criticality Detector_Negative Feedback Circuit
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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!
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part: Pompc+taRNA+B0010
Colony pcr using primer VF2 and VR
Theoretical length (from VF2 to VR) : 598 bp
Construction succeeded!
Dosage Control System
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Colony pcr
part: APP2 (48bp)
part: Bac8c (30)
part: CAP (28bp)
part: CAP-app2(82bp)
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.
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.2 Efficient insertion requires the first step.
Fig.1 The first pair of primers "open" the circular plasmid at a specific cite.
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.2 Efficient insertion requires the first step.
CI Binding Site Insertion_A Brand-new PCR Strategy
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On the left, most colonies were red indicating the aborted insertion by the one-step PCR (without step1); on the right, most colonies lost their color for the successful insertion by the two-step PCR.
Step2:The second pair of primers is for insertion of the DNA segment and back-splicing of the linear plasmid. The pair of primers not only respectively overlaps with both terminus of the linear product of the first step, but also overlaps with each other to back splice the linear plasmid. Thus the insertion is completed and the product of the reaction becomes circular plasmids ready to be transformed into competent cells.
It is noteworthy that the second pair of primers in our PCR strategy doesn’t need to be complementary to each other. The gap shown below (see Fig.4) could be filled by DNA polymerase and therefore, the length of both the forward and reverse primer could be reduced to nearly one-half of the DNA segment for insertion. In such a way, price for primers synthesis could be reduced too.
Fig.4 Only a small overlap is adequate for the second pair of primers.
Fig.4 Only a small overlap is adequate for the second pair of primers.
Application: Targeted Antimicrobial Peptides AAP2&Bac8c
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Curing through synthetic biology has been a hot issue for a long time. Since one of our team mem-ber was troubled by dental caries since very young, and since our criticality detector can be used to control doses of some strong medicine precisely by receiving complicated input signal and transfer it into a simple one. So to prove this property creatively, we construct an antimicrobial peptide pro-ducing circuit which is helpful for dental caries prevention and homeostasis maintenance.
To be specific, when the concentration of Streptococcus mutans increase to a certain level, the criticality detector can activate the expression of targeted antimicrobial peptide to inhibit the growth of S.mutans, controlling its population in oral cavity under a healthy level.
Fig.4 Only a small overlap is adequate for the second pair of primers.
Fig.4 Only a small overlap is adequate for the second pair of primers.
Mechanism of our Targeted antimicrobial peptides
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Antimicrobial peptides are recently regarded as a promising choice to kill microbes due to their strong microbicidal effects and their unlikeliness of triggering resistance in microorganisms. Since there are also various kinds of probiotics living in our mouth besides oral pathogens, and since antimicrobial peptides have effect on general kinds of microorganisms, it’s more wise to modify the antimicrobial peptide into a targeted one by adding a S.mutans targeted sequence on the N-terminal, so that only S.mutans can be killed selectively. Thus we design two fused peptides, CAP-glyglygly-Bac8c and CAP-glyglygly-AAP2, to targetedly inhibit the growth of S.mutans in oral cavity, where CAP is an optimized peptide that can specifically bind to the comD receptor on the surface of S.mutans, while Bac8c and AAP2 are two kinds of peptides that assume strong bactericidal effect in acidic environment.
Descriptions of our plasmids
The 5 plasmids show below are the standard parts we’ve registered on time:
Application: Targeted Antimicrobial Peptides AAP2&Bac8c
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Then after a series of molecular biological experiments, we finally constructed 3 function proving plasmids (show below) and registered our standard parts on time.
For more information of our parts and application programs please checkhere
Then we transformed these plasmid into the E.coli (BL21) to test the expression and function of our
antimicrobial peptides, and thus conduct 3 groups of function testing experiments as follow:
Then we transformed these plasmid into the E.coli (BL21) to test the expression and function of our
antimicrobial peptides, and thus conduct 3 groups of function testing experiments as follow:
Function Testing Experiments
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1.Turbidimetry
To test the activity of AAP2 and Bac8c under different pH conditions, we conducted 2 experiments below with a 722s spectrometer.We cultured the two kinds of E.coil (BL21), one containing the plasmid Pet28a-AAP2 and the other containing the plasmid Pet28a-Bac8c, in the liquid Luria-Bertani media.
A. Effect of AAP2 on E.coli under different pH
After being cultured to OD=600, E.coli containing Pet28a-AAP2 are induced with 1.0mM IPTG and the pH of the bacterial solution is adjusted to either pH=5 or pH=7 by adding hydrochloric acid. Then the OD of the solution is analyzed every 30 minutes, and the result are as follows:
From the picture we can see that with the induction of IPTG, E.coli's growth is strongly inhibited af-ter 5.5 hours, indicating that AAP2 is an effective antimicrobial peptide. However, the inhibition ef-fect of AAP2 are similar between groups pH7 and pH5, indicating that AAP2 may not be an acid activated antimicrobial peptide.
Nevertheless, considering that the pH of the external environment is much easier to change than that of the inner cell environment (because of the homeostasis of E.coli), and since AAP2, after in-duction, is expressed and takes effects inside E.coli, we can't say that the pH change of the exter-nal environment will indeed affect the structure of AAP2 inside E.coli, and thus we can't deny it that AAP2 may have stronger inhibiting effect at lower pH. Further experiment should be conducted to prove our guess, where the expressed AAP2 will be purified and artificially added to an E.coli cul-ture, so that the AAP2 can take effect from the outside of the cell.
However, according to the result we get, it's one thing for sure that AAP2 has antimicrobial effect under pH7.
B. Effect of Bac8c on E.coli under pH=7.0
After being cultured to OD=600, E.coli containing Pet28a-AAP2 are induced with 1.0mM IPTG and the pH of the bacterial solution is adjusted to either pH=5 or pH=7 by adding hydrochloric acid. Then the OD of the solution is analyzed every 30 minutes, and the result are as follows:
From the picture we can see that with the induction of IPTG, E.coli's growth is strongly inhibited af-ter 5.5 hours, indicating that AAP2 is an effective antimicrobial peptide. However, the inhibition ef-fect of AAP2 are similar between groups pH7 and pH5, indicating that AAP2 may not be an acid activated antimicrobial peptide.
Nevertheless, considering that the pH of the external environment is much easier to change than that of the inner cell environment (because of the homeostasis of E.coli), and since AAP2, after in-duction, is expressed and takes effects inside E.coli, we can't say that the pH change of the exter-nal environment will indeed affect the structure of AAP2 inside E.coli, and thus we can't deny it that AAP2 may have stronger inhibiting effect at lower pH. Further experiment should be conducted to prove our guess, where the expressed AAP2 will be purified and artificially added to an E.coli cul-ture, so that the AAP2 can take effect from the outside of the cell.
However, according to the result we get, it's one thing for sure that AAP2 has antimicrobial effect under pH7.
B. Effect of Bac8c on E.coli under pH=7.0
Function Testing Experiments
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After being cultured to OD=600, E.coli containing Pet28a-Bac8c are induced with 1.0mM IPTG and the OD of the solution is analyzed at 0, 1, 3, 7 hours after induction. The result are as follows:
From the picture we can see that with the induction of IPTG, E.coli’s growth is strongly inhibited af-ter 7 hours, indicating that Bac8c is an effective antimicrobial peptide.
2. SDS-PAGE Electrophoresis
To detect the antimicrobial peptides expressed by E.coli (BL21), we collected 70mL sample from each group of the Turbidimetry experiment above after inuction for 240min. Then we lysed the samples with an ultrasonic Processor, getting 3 kinds of bacterial lysis solutions:
Pet28a-AAP2, pH=7.Pet28a-Bac8c, pH=7Pet28a-CAP-Bac8c, pH=7
With the 12% separating gel and the 3% stacking gel;we had our samples with loading buffer run for 20min in stacking gel under 120V and 60min in separating gel under 100V.
As in the picture, the band of the three antimicrobial peptide AAP2, Bac8c and CAP-Bac8c are really vague and unclear,even we cannot give a label. However the samples were also tested for the Kirby–Bauer antibiotic testing, which confirmed the existence and the function of our peptides. So here are several possible reasons:
The relative molecular mass of those peptides are no more than 2kD, which is very difficult to dis-tinguish on the gel. Besides the band intensity of other proteins are very light, indicating that the concentration of our sample may be very low, making it even harder to identify the band of antimi-crobial peptide.
From the picture we can see that with the induction of IPTG, E.coli’s growth is strongly inhibited af-ter 7 hours, indicating that Bac8c is an effective antimicrobial peptide.
2. SDS-PAGE Electrophoresis
To detect the antimicrobial peptides expressed by E.coli (BL21), we collected 70mL sample from each group of the Turbidimetry experiment above after inuction for 240min. Then we lysed the samples with an ultrasonic Processor, getting 3 kinds of bacterial lysis solutions:
Pet28a-AAP2, pH=7.Pet28a-Bac8c, pH=7Pet28a-CAP-Bac8c, pH=7
With the 12% separating gel and the 3% stacking gel;we had our samples with loading buffer run for 20min in stacking gel under 120V and 60min in separating gel under 100V.
As in the picture, the band of the three antimicrobial peptide AAP2, Bac8c and CAP-Bac8c are really vague and unclear,even we cannot give a label. However the samples were also tested for the Kirby–Bauer antibiotic testing, which confirmed the existence and the function of our peptides. So here are several possible reasons:
The relative molecular mass of those peptides are no more than 2kD, which is very difficult to dis-tinguish on the gel. Besides the band intensity of other proteins are very light, indicating that the concentration of our sample may be very low, making it even harder to identify the band of antimi-crobial peptide.
Function Testing Experiments
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3.Kirby–Bauer antibiotic testing
To confirm the existence and the function of our antimicrobial peptides, we conducted the experiment using the bacterial lysis solutions we had collected earlier :
Using an aseptic technique, place a sterile glass rod onto the culture of E.coli and then gently streak the Mueller-Hinton agar plate to form a bacterial lawn.To obtain uniform growth, streak the plate with the glass rod to and fro, meanwhile rotate the plate 90° and streak the plate again and again.
Allow the plate to dry for approximately 5 minutes.
Using an Antibiotic Disc Dispenser to dispense discs containing our antimicrobial peptide and also sterile water onto different plates.
Using a flame-sterilized forceps, gently press each disc to the agar to ensure that the disc is at-tached to the agar.
Plates should be incubated overnight at an incubation temperature of 37 °C for 16 hours.
Observing and comparing the zones of inhibition in different plates.
A.pET-AAP2 (pH=5) B.pET-Bac8c (pH=5) C.75% ethanol (left)/ sterile water (right) D.pET- AAP2 (pH=7) E.pET-CAP-Bac8c (pH=7)
This picture shows different kind of antimicrobial peptide we designed that were used in Kirby–Bauer antibi-otic testing. Through this, we could find that comparing with negative control (sterile water) in plate C, even not that clearly, the diameters of zones of inhibition are larger. Because the antibacterial effect of ethanol is not prominent, the diameter in plate C are small.
Comparing plate A with D, since AAP2 should have stronger effect under low pH, and the results show that the diameter in A are larger than D, indicating that under low pH environment the AAP2 are more effective.
Comparing plate B with E, we test whether CAP may have influence on the bacteriostasis of Bac8c. Based on our assumption, CAP have a special function on pinpointing Streptococcus. mutans, and the fusion of CAP cannot interfere Bac8c. This picture shows that the existence of CAP do not influence the diameter of the zone of inhibition, proving our assumption.
Despite the fact that our experiment may have some flaws, we can still make a simple conclusion that these antimicrobial peptide indeed have bacteriostasis, and they can work under different con-ditions.
A.pET-AAP2 (pH=5) B.pET-Bac8c (pH=5) C.75% ethanol (left)/ sterile water (right) D.pET- AAP2 (pH=7) E.pET-CAP-Bac8c (pH=7)
This picture shows different kind of antimicrobial peptide we designed that were used in Kirby–Bauer antibi-otic testing. Through this, we could find that comparing with negative control (sterile water) in plate C, even not that clearly, the diameters of zones of inhibition are larger. Because the antibacterial effect of ethanol is not prominent, the diameter in plate C are small.
Comparing plate A with D, since AAP2 should have stronger effect under low pH, and the results show that the diameter in A are larger than D, indicating that under low pH environment the AAP2 are more effective.
Comparing plate B with E, we test whether CAP may have influence on the bacteriostasis of Bac8c. Based on our assumption, CAP have a special function on pinpointing Streptococcus. mutans, and the fusion of CAP cannot interfere Bac8c. This picture shows that the existence of CAP do not influence the diameter of the zone of inhibition, proving our assumption.
Despite the fact that our experiment may have some flaws, we can still make a simple conclusion that these antimicrobial peptide indeed have bacteriostasis, and they can work under different con-ditions.
Conclusions
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We tested the function of our antimicrobial peptides in E. coli through Turbidimetry, SDS-PAGE Electrophoresis and Kirby–Bauer antibiotic testing. Based on the result we can confidently conclude that:
• All the peptides can be expressed correctly in E.coli(BL21);
All the peptides have expected bacteriostasis function in E.coli;
Bac8c is more active under pH=7 while AAP2 is more active under pH=5;
The peptides fused with CAP guiding part have stronger inhibiting effect.
