Team:BroadRun-NorthernVA/Methods
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Methods
To genetically engineer a yeast cell to produce and secrete alpha amylase, the process of inserting a gene of interest into a cloning vector was used.
The image below provides an overview of the cloning process.
Three different composite gene constructs were designed, each with a alpha amylase coding sequence from B.amyloliquefaciens. In each of the constructs, we tried different combinations of promoters and secretion sequences.
Construct 1
Construct 2
Construct 3
The first construct was promoterless with the native secretion sequence, the second used a cyc promoter and the native secretion sequence of the alpha amylase gene, and the third used a cyc promoter and the native secretion sequence was replaced with a mating factor secretion sequence.
The gene constructs sequences were optimized for S.cerevisiae and restriction enzyme sites were removed.
Integrated DNA Technologies then synthesized our gene constructs and the accompanying primer sets for each.
Amplification
The gene constructs were amplified using Polymerase Chain Reaction.
The PCR products were purified with a spin kit and the restriction digests set up. The three plasmids were also digested; pSB1C3, pRS426, and pAG36. pSB1C3 and pRS426 were both digested with EcoR1 and Pst1, and the pAG36 was digested with Kpn1 and Spe1. All three constructs were all digested with EcoR1 and Pst1, and in a separate reaction, construct 3 was digested with Kpn1 and Spe1. After heat inactivation at 80 Celsius, we used gel extraction to ensure we had the right fragments.
All three constructs were ligated with the pSB1C3 plasmid. Constructs 1 and 2, the two with promoters, were ligated into the pRS426 yeast vector, which does not contain a promoter. Construct 3, which does not have a promoter, was ligated into the pAG36 vector, which contains a TEF1 promoter. The TEF1 promoter is also a constitutive promoter. The ligation reactions were transformed into E.coli bacteria and then plated.
After growing cultures overnight, three colonies were chosen from each plate for colony PCR, streaked onto master plates, and inoculated in liquid cultures. After colony PCR confirmed which colonies had the correct insert, those colonies were miniprepped after the liquid cultures had grown overnight. The DNA samples with the pAG36 and pRS426 vectors were transformed into yeast cells and grown for two days before testing.
The gel to the left shows the results for each of the three constructs after amplification PCR. The 1kb ladder confirms the constructs are the correct size, about 2kb. Below, Dr.Scheifele points out the distinct bands on the gel.
Restriction Digest
The gel above shows the two distinct fragments after restriction digest for each of the three plasmids, pSB1C3, pRS426, and pAG36. A gel extraction was performed on the higher bands of DNA.
Ligation
The master culture plates after bacterial transformation and Colony PCR.
Colony PCR
The image to the left shows the colony PCR results. Below, Dr.Burkett and Marissa discuss and inspect the bacterial colonies on an agar plate.
