Our project aims to develop a disinfectant hydrogel plaster with a long-lasting sterilizing effect. We address the challenges posed by bacterial infections. After modeling part, seven candidates (CLP1, CLP2, CLP3, DLP1, DLP2, DLP3 and DLP4) are considered as potential effective antimicrobial peptides (AMPs). We have designed eight BioBrick™ to express seven AMPs in our expression system. T7-His tag-SUMO tag-intein-defensin-like peptide 4(DLP4) [BBa_K4794008] includes T7 promoter [BBa_I719005] for the start of expression, His-tag [BBa_K128005] for purification, SUMO-tag [BBa_K2342001] for the increase of expression rate and solubility of AMPs, intein to inhibit the AMPs during expression and AMPs itself. [BBa_K4794000] is the intein-DLP4. [BBa_K4794001] - [BBa_K4794006] and [BBa_K47940011] are DLP1-3, CLP1-3 and DLP4 respectively.
(i) Digestion, ligation and transformatoin
SalI and KpnI were used to cut open the pET plasmid and also the intein-DLP4 insert and then both fragments were ligated. The diagram below shows that intein-DLP4 carried by the plasmid provided by IDT company are successfully digested by SalI and KpnI. Target bands with 620bp (intein-DLP4) are observed, so the target gene is cut successfully.
Then, SacI and KpnI were used to cut open the pET plasmid-interin-DLP4 and also the CLP1-3 and DLP1-3, total six inserts and then both fragments were ligated to form six plasmids with different AMP inserts. The diagram below shows that all inserts and the pET plasmid are successfully cut by SacI and KpnI.
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(ii) Colony PCR and double digestion
Total six recombinant plasmids are transformed into BL21 and colonies were observed in LB plate with kanamycin. For the pET plasmid-intein-CLP2, there are few colonies, so we skip colony PCR and driectly checked by double digestion. For the others, colony PCR were carried out and each plates get +ve results in some colonies. Below shows the results:
After colony PCR, +ve colonies were picked, culture and miniprep. And then the miniprep products would be digested by different combination of restriction enzymes. For each AMPs, at least one sample shows target band. That can further confirm a correct recombinant plasmid was transformed in BL21. Below show the results:
(i) IPTG induction
The constructed expression vector was inserted into BL21 for expression. The protein was induced with 0.4mM IPTG for 24hrs at 20℃. And then, the cells were harvested by centrifugation at 6000xg for 20mins. After induction, SDS-PAGE has been conducted to check whether the protein has been successfully induced. 1 ml Lysis buffer with 1mM PMSF per 10mL culture was used to resuspend the pellet. Suspension was sonicated on ice (Amplitude: 95%; Time 5 min; Pulse 2 son, 2 s off). It was cleared by centrifugation at 150000 rpm, 4℃for 15 min. Protein band at about 50 kDa was observed in both supernatant (S/N) and pellet for CLP1 and DLP1-4 before purification. The results don’t fully match with our expectation. First, CLP2 and CLP3 fail in expression. And for the others, there are quite lots of insoluble target proteins in pellet, although AMPs are water-soluble in nature and SUMO-tag should increase the solubility.
(ii) Purification
Suspension of CLP1 and DLP1-4 were purified by Ni-NTA resin and eluted in PBS. And then, SDS-PAGE has been conducted to estimate the concentration of AMPs. Protein band at about 50 kDa was observed in elution for DLP1-3 after purification but the concentrations were too low for further study. For CLP1 and DLP4, Protein band at about 50 kDa was observed in elution for both AMPs after purification. The concentration of CLP1 and DLP4 were further measured by nanodrop, 0.582 mg/mL and 0.961 mg/mL respectively. These two AMPs were selected for cleavage and antimicrobial assay.
(iii) Intein self-cleavage
Self-cleavage of purified CLP1 and DLP4 was performed in 20mM phosphate buffer, pH10 at 55℃ for 72hours. The set-up has shown below.
After cleavage, SDS-PAGE has been conducted. Protein band at about 15 kDa was observed in cleavage. while band at about 50kDa was still presence, which shown the cleavage was partially completed for both CLP1 and DLP4.
(i) General description
Clear zones have been observed in all cleaved DLP4, CLP1 and positive control. No clear zone can be observed in all uncleaved DLP4, CLP1 and negative control. Compared to DLP4 and CLP1, ampicillin has the largest clear zone, showing stronger antimicrobial effect. And also, the lower the concentration of DLP4 and CLP1 are, the smaller the clear zones are. This shows proportional relationship of antimicrobial effect of AMPs and the concentration of AMPs. In conclusion, preliminary results show AMPs obviouly shows antimicrobial effect on Lactobacillus spp., a gram positive bacteria. The effect of DLP4 and CLP1 are similar, and both of them have weaker effects than ampicillin.
(ii) Limitations
First, we don’t have enough time to purify strain of Lactobacillus spp. and we have just poured concentrated yogurt drink [Yakult] on MRS agar. As, the solution of yogurt drink is so sticky, we cannot spread evenly on the agar plate. This may affect the accuracy of the results, like the negative control of agar plate of cleaved CLP1 [diagram 20]. There are no colonies formed near the disc with distilled water. However, the zone is not circular, we don’t count it as ‘clear zone’. Second, there is hugh loss of AMPs after the self-cleavage. One of the possible reasons maybe the temperature is not optimal, so the self-cleavage cannot completed.
(iii) Imporvement
In the future, repeat is required to control the initial bacterial culture concentration and the purity of the strain. Also, we should figure out the optimal conditions of intein self-cleavage to increase the concentration of AMPs collected.
(i) Production
Our antimicrobial BSF plaster has been successfully made by the procedures listed in experiment part. The plaster made contain two layers. The outmost layer is roasted in order to make it to be waterproof. The inner layer doesn’t be roasted, so it can be soft and more comfortable.
(ii) Biodegradability
BSF plaster took 45 days for complete biodegradation. Obviously, anti-bacterial and non-allergic BSF plaster were biodegradable. On the other hand, the mass of commercial plaster decreased only by 51.1% in 45 days, which was much slower.
(i) Successes
1. Seven types of recombinant plasmids have been made.
2. Seven types of plasmids have been transformed into BL21.
3. CLP1, DLP1-4, total five AMPs have been induced..
4. Enough high concentration of intein-CLP1 and intein-DLP4 have been purified.
5. Antimicrobial test has been done to prove the antimicrobial effect of CLP1 and DLP4.
6. Antimicrobial BSF plaster has been made.
7. Biodegradability test has been done to prove that degradation rate of BSF plaster is faster than ordinary plaster.
(ii) Failures
1. CLP2 and CLP3 were failed to be induced.
2. The concentration of purified DLP1-3 are too low.
3. The efficiency of intein self-cleavage is low.
We found that in AMPs were found in both S/N and pellet, but not all in S/N. We have to design some new composite parts to solve this problem. Another two new composites have been designed for the possible solution for the problem of low solubility of AMPs. For the first one, SUMO-tag is simply deleted, T7-His tag-intein-AMP [BBa_K4794009], to check whether the solubility can be increased by reducing the size of protein synthesized. For another composite, SUMO tag is replaced by ELK-16 system, T7-AMP-intein-PT linker-ELK16 [BBa_K4794010] to change the purification mechanism by changing the solubility of AMP from insoluble to soluble form[1].
Also, we should figure out the optimal conditions for intein self-cleavage to increase the efficiency. And then, more repeat tests should be conducted to increase the accuracy of the antimicrobial test and should control the initial bacterial culture concentration by purifying the strain.
Moreover, we have to do more tests to find out the working conditions for AMP and how to fix the AMP in our plaster for better and long-lasting antimicrobial effect.
Wang, M., Zheng, K., Lin, J., Huang, M., Ma, Y., Li, S., Luo, X., and Wang, J. (2018). Rapid and efficient production of cecropin A antibacterial peptide in Escherichia coli by fusion with a self-aggregating protein. BMC Biotechnol. 18, 62