Results
Below shows the sizes of the DNA fragments:
| 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 |
PCR
PCR was used to replicate DNAs. Below are the gel photos showing the result of PCR.
Figure 1a: 1%agarose gel showing the results of PCR (the bands of masp2_2(947bp), prir2(533bp), masp2_1(908bp), masp1_102(935bp), piri4(911bp), tusp(1157bp), masp1_1(1253bp))
Figure 1b: 1%agarose gel showing the results of PCR (the band of piri6(1337bp))
Figure 1c: 1%agarose gel showing the results of PCR (the band of piri5(860bp))
As shown in the figures, all genes were replicated successfully except Piri5 and Masp1_103.
Digestion
Restriction enzymes sacI and salI were used to digest the inserts and backbone. The bands of successfully digested DNA fragments should travel slightly faster than that of undigested DNA fragments.
Same as before, the genes were extracted via gene clean and stored at -20°C
Figure 2a: 1%agarose gel showing the results of digestion (the bands of piri6, tusp and masp1_1)
Figure 2b: 1% agarose gel showing the results of digestion using SalI and SacI (masp2_2, prir2, masp2_1)
As shown on the figure, piri6 , tusp and masp1_1, masp2_2, prir2, masp2_1 were digested successfully and extracted via gene clean.
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.
Figure 3a: 1%agarose gel showing the results of colony PCR. 6 colonies were picked for piri6. Correct band sizes and location were labelled.
Figure 3b: 1%agarose gel showing the results of colony PCR. 8 colonies were picked for tusp. Correct band sizes and location were labelled.
Figure 3c: 1%agarose gel showing the results of colony PCR. 8 colonies were picked for tusp. Correct band sizes and location were labelled.
Re-digestion
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.
Figure 4a: 1%agarose gel showing the results of digestion of plasmid obtained from the colonies having +ve results in colony PCR. Correct band sizes and location were labelled.
Figure 4b: 1%agarose gel showing the results of digestion of plasmid obtained from the colonies having +ve results in colony PCR. Correct band sizes and location were labelled.
As shown in figures,the recombinant plasmids that inserted Piri6 and tusp are matched the expectations. They were sent to sequencing company to check the insert integrity to ensure spidoin produced will have the expected amino acid sequence.
Sequencing
The sequencing results were returned in the form of .ab1 files. They were then imported into Benchling for alignment with the expected sequence to check if there are any mutations detected. Comparisons have shown that no mutations are found in some of the samples. They will then be used for protein expression.
Figure 5a: Sequence alignment for TuSp
Figure 5b: Sequence alignment for Piri6