Team:Jianhua/Engineering
Engineering
Parts Submitted
Basic Parts:
| Part Name | Type |
|---|---|
| BBa_K4081992 | FKP |
| BBa_K4081996 | Lac12 |
| BBa_K4081998 | FucT2 |
| BBa_K4081995 | TEF1 promoter |
| BBa_K4081994 | GAP promoter |
| BBa_K4081990 | ADH1 promoter |
| BBa_K4081888 | CDT2 |
Composite Parts:
| Part Name | Type |
|---|---|
| BBa_K4081085 | pGAP-FKP-tADH1 |
| BBa_K4081238 | pTEF1-Lac12-tADH1 |
| BBa_K4081333 | pADH1-FucT2-tADH1 |
abstract
This year, we successfully co-express three proteins(FKP, LAC12, FucT2) in Saccharomyces cerevisiae BY4741. These three genes will work together to synthesize 2’-FL.
Construct recombinant plasmids
1.We PCR GAP promoter(BBa_K4081994) from vector PML104. Get TEF1 promoter (BBa_K4081995) from the genome of Saccharomyces cerevisiae BY4741. Get ADH1 promoter(BBa_K4081990) from vector pAUR123.
Figure 1. Gel electrophoresis results of PCR promoter. Lane 1: Marker; Lane 2: GAP promoter(700bp); Lane 3: TEF1 promoter(577bp); Lane 4: ADH1 promoter(461bp).
2.The result of PCR showed that we successfully construct recombinant plasmid.
Figure 2. Restriction digest test result proves success.
3.To get the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene AurR from the plasmid with homology arms of BY4741, we perform PCR twice, and then connect the two fragments with ligase. The gel electrophoresis results of PCR shows we successfully get the target fragment.
Figure 3. PCR the target fragment: the genes “pGAP-FKP-pADH1-FucT2-pTEF1-LAC12” and the resistance gene “AurR”. Line1: Marker; Line2-6: target fragment(11022bp).
Co-express three proteins(FKP, LAC12, FucT2)
1.Transform the target fragment into BY4741 by lithium acetate conversion method to integrate genes into the genome of BY4741 for expression. Screen for transformants by AbA-YPD selection medium.
Figure 4. Transformants on AbA-YPD selection medium
2.The result of PCR showed that we successfully integrated the target fragment into the yeast genome.
Figure 5. Gel electrophoresis results of PCR. Lane 1: Marker; Lane 2: PCR product of the upstream of integration site (1797bp); Lane 3: PCR production of the downstream of integration site (1005bp).
3.The result of SDS-PAGE showed that we successfully co-express three proteins FKP(BBa_K4081992), LAC12(BBa_K4081996) and FucT2(BBa_K4081998).
Figure 6. The result of SDS-PAGE. Line 1: Marker; line 2: Control; line 3: Express FKP(106KD), LAC12(65KD), FucT2(35KD).
To conclude, we demonstrated the engineering success of our modules through experiments.