After modeling part,seven candidates(CLP1,CLP2,CLP3,DLP1,DLP2,DLP3 and DLP4) are considered as potential effective AMPs.These seven genes were inserted into out pET plasmid and transformed into BL21.After purification,these seven AMPs were tested and we compared their effectiveness in gram-positive and gram-negative bacteria.
The plasmid we designed contains an insert of seven AMPs respectively, and ligated with the pET plasmid. The pET plasmid contains kanamycin resistance gene, SUMO-tag and a Multiple Cloning Site. Apart from the AMP gene, Polyhistidine-tag (6-His tag) and intein are also included in the insert. Moreover, SalI and KpnI, two restriction sites are designed on both ends of insert for insert-vector ligation. Also, SacI, another restriction site is added between AMP and intein, so that the AMP can be replaced with another easily.
Figure 1:Overview of plasmid design.The insert is DLP4
It is the most frequently used as N-end fusion sequence to increase the expression and solubility of the desired recombinant protein. Attachment can be removed by SUMO-specific isopeptidases just as UCHs and UBPs remove ubiquitin from substrate proteins.
A region of DNA within a plasmid. It contains multiple unique and contiguous restriction enzyme recognition sites, so it provides flexibility in the choice of restriction enzymes. In our plasmid design, the 3 cut sites (SalI, SacI and KpnI) are included. SalI and KpnI, which is located at both ends of intein-DLP4 and are used for the assembly of intein-DLP4 to pET plasmid. SacI is located between intein and DLP4 and are used with kpnI for the change of another AMPs.
Figure 2(a):intein-DLP4 insert design
Figure 2(b):Other AMPs insert design
It is one of the simplest and most widely used purification tags and commonly used in the production of recombinant proteins. These residues readily coordinate with transition metal ions immobilized on beads or a resin for purification.
It inactivates the antimicrobial peptide so that it will not kill the bacteria during expression. This can ensure continuous expression of AMPs. After protein purification, the intein would be removed by autocleavage by excising itself in pH 10 buffers.
Table 1:Nine BioBrick™ designated by our team
Before digestion and ligation, all plasmids are amplified by DH5-alpha bacterial culture and purified by miniprep. Next, SalI and KpnI were used to cut open the pET plasmid and also the intein-DLP4 insert and then both fragments were ligated.
The recombinant plasmid first was transformed in DH5-alpha for cloning. And then, the plasmid was transformed in BL21 after the ‘Part C: Selection’.
In order to confirm whether the bacteria in colony get the correct plasmid, colony PCR was conducted. We designed some primers for colony PCR. Details show below.
Table 2:Sequence of primers for colony PCR
For those colonies who get positive results in colony PCR, they were picked up for further culture and the plasmids were purified by miniprep. Next, SacI and KpnI were used to digest the plasmid and then gel electrophoresis was carried out. If the target band appears, the corresponding plasmid will be transformed in BL21 for later expression. After the transformation, ‘Double digestion’ would be conducted again to confirm the BL21 colony really get the correct plasmid.
SacI and KpnI were used to digest the pET plasmid-DLP4 and other AMP inserts and both fragments were ligated to form different pET plasmid-six AMPs respectively. We then repeated Part B: transformation and Part C: Selection again for those six recombinant plasmids.
A single colony was picked up and incubated in 20ml LB broth containing kanamycin(1X) at 37℃ with 150rpm shaking overnight. A total of 6ml incubated cell culture was then incubated into 240ml LB broth containing kanamycin(1X) at 37℃ with 150rpm shaking until the OD600 reached 0.6. Then, 0.4mM IPTG was used to induce the protein expression for 24hrs at 20℃. This step repeated seven times for seven AMPs expression.
SDS-PAGE would be conducted to test did the expression be induced successfully and estimate the protein concentration.
If target bands have been shown in SDS-PAGE results, the corresponding supernatant samples will undergo his tag purification. After that, another SDS-PAGE will be conducted to confirm whether the AMPs were purified.
Self-cleavage of purified His6-intein-AMPs was preformed in 20mM phosphate buffer, pH 10 at 55℃ for 72hrs. Afterward, AMP was precipitated in the pellet. The, the AMP was obtained by centrifugation (12,500xg, 30mins, 4℃). The AMP was further resuspended by 6M guanidine hydrochloride.
Antimicrobial test was held to check the antimicrobial property of the AMPs.
First, an agar plate was spread evenly with Lactobacillus spp.
Second, five paper discs, each of which was soaked with the different concentration of AMPs, antibiotics or distilled water, were placed on the agar.
Third, the plate was incubated at 37℃ for 24-48hrs to allow the growth of the bacteria.
Last, the diameters of the clear zones formed around the discs were measured using a ruler.
Figure 3: Antimicrobial test procedures
Table 3:Evaluation of inhibition of bacteria
Hydrogel of chitosan from Black Soldier Fly (BSF) is anti-bacterial (with the presence of AMP), non-allergic and biodegradable. After modification, it would become waterproof. However, commercial plaster such as hydrocolloid are neither biodegradable nor anti-bacterial. Therefore, the biodegradability of BSF plaster makes it environmentally friendly, which takes the responsibility of sustainable consumption and production of the 17 Sustainable Development Goals from United Nation.
First,demineralization and deproteinization of BSF shells in 2M HNO3.
Then,deacetylation was done by adding excess 16.7M NaOH to the BSF chitin samples
Last,production of BSF hydrogel using excess acetic acid
BSF plaster and commercial plaster were buried in soil and their mass were measured regularly to see the degradation conditions.