Part:BBa_K4000000

TEF1p

TEF1p

Contribution By Team iGEM23_SubCat-China

Group: SubCat-China 2023

Summary: Based on BBa_K4000000 (TEF1 promoter), we add the part BBa _ K4845004 (GAP promoter), BBa _ K4000002 (CYC1 terminator), BBa _ K4845007 (ADH1 terminator), BBa _ K4845009 (temA) to BBa _ K4845003 (X-3-backbone). The new recombinant plasmid BBa _K4845017 (X-3-temA-2) was constructed to increase two promoters and terminators, two temA on the X-3-backbone plasmid, improve the enzyme activity of glucoamylase, and further improve the ability of yeast to decompose starch.

a. Usage and Biology

In alcoholic fermentation, α-amylase cannot hydrolyze α-1,6 glycosidic bonds. The complete hydrolysis of starch requires the synergistic effect of α-amylase and glucoamylase, but α-amylase is considered to be more important than glucoamylase because the hydrolysis of starch into oligosaccharides by α-amylase may be the rate-limiting step. Therefore, the glucoamylase was integrated into the plasmid, and the starch α-1,4 glycosidic bond was rapidly hydrolyzed, and the α-1,6 glycosidic bond and α-1,3 glycosidic bond were slowly hydrolyzed, and the final product was all glucose.

b. Characterization/Measurement

In order to construct BBa _K4845017 (X-3-temA-2), we firstly amplified the GAP, TEF1 promoters, temA key genes and CYC1, ADH1 terminators through PCR(Table 1). Since we would insert two temA genes into the same plasmid, we used two different promoters and two different terminators. With preparation of basic materials, we linked the promoters, key genes and terminators together through Over PCR to construct GAP-temA-CYC1 and TEF1-temA-ADH1 genes which will be inserted into the plasmid skeletons (X-3) later (Figure 1). What is more, the design of our target genes is displayed both in the form of table and the form of visualized diagram.

Figure 1: Visualized blocks-assembly diagram for showing our design of temA DNA template
Upper one: GAP-temA-CYC1 gene of 2804 bp
Lower one: TEF1-temA-CYC1 gene of 2482 bp

After we obtained our target temA gene fragments, we inserted them into X-3 plasmid skeletons through restriction endonuclease digestion and ligation method (Figure 2).

Figure 2: Visualized models of our plasmids designed (X-3-2temA)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 189
  Illegal SapI site found at 3
  Illegal SapI.rc site found at 426