Team:HK CPU-WFN-WYY/Experiment


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Experimental design

Workflow

Plasmid design

Our plasmid consists of 3 parts: the origin of replication, kanamycin resistance gene and the expression system. The origin of replication is needed for the propagation of the plasmid in E. coli. The kanamycin resistance gene gives transformants resistance to kanamycin for selection purposes. The expression system composes of an inducible promoter, restriction sites, the gene of interest and the terminator. The inducible promotor and the terminator combined allows for the regulation of the gene of interest using IPTG as the inducer. The restriction sites are used to clone the gene of interest into the plasmid backbone. The gene of interest encodes the spidroin that we want to express. The expression system expresses the spidroin with a his-tag (not shown in the diagram) which helps in the isolation and purification of the desired spidroin.

PCR

In this process, PCR was used to replicate DNAs, including Piri6, Masp2_2, Piri2, Masp2_1, Piri5, Masp1_102, Piri4, Tusp, Masp1_1.

After PCR, gel electrophoresis was performed to ensure the PCR products have expected sequences. The genes which had expected bands were extracted through gene clean and stored at -20°C.

Gene whole gene size (bp)
Piri6 1337
Masp2_2 947
Piri2 533
Masp2_1 908
Piri5 860
Masp1_102 935
Piri4 911
Tusp 1157
Masp1_1 1253

Digestion

Restriction enzymes SacI and SalI were used to digest the inserts and backbone in order to create complementary sticky ends for ligation. Before carrying out the ligation process, gel electrophoresis was performed to ensure the inserts were digested.

Same as before, the genes were extracted via gene clean and stored at -20°C

Ligation

The complementary sticky ends on the backbone and inserts were ligated to create the desired recombinant plasmid.

Transformation

Before transformation, the competent cells were prepared by mixing the cultured E.coli DH5a ,calcium chloride and calcium chloride with glycerol in different processes. This made the cells become positively charged so that they were easier to take up plasmids which were negatively charged. Since the competent cells were highly unstable, they were stored at -80°C before use.

The ligation products and chemically competent cells were ligated and undergone heat shock. This helped the cells take up recombinant plasmids.

After that, recovered and plated the cells on the LB agar plate with kanamycin. Only the E.coli that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

Colony PCR

After incubation, the colonies were picked to perform colony PCR in order to differentiate between undigested plasmid transformants and recombinant plasmids transformants.

The colonies with the correct band size were extracted from the plate and cultured by liquid culture in order to get a large amount of plasmids for further analysis.

Miniprep

Miniprep was performed to extract the recombinant plasmids from the cultured recombinant E.coli.

Re-digestion

In order to differentiate between the self-ligated plasmids and recombinant plasmids which contain target genes, re-digestion was carried out. The miniprep products were digested by restriction enzymes sacI and salI and then ran gel to check the bands. Since the target gene and plasmids were cut and separated, there should have 2 bands in gel electrophoresis, one was the band of plasmid, which is around 5000bp, and one was the band of target gene, the size depended on the type of genes.