Part:BBa_K4845017
X-3-temA-2
X-3-temA-2
Composite Part BBa_K4845017 (X-3-temA-2)
Construction Design
Based on BBa_K4000000 (TEF1 promoter), we added the following parts:
- BBa_K4845004 (GAP promoter)
- BBa_K4000002 (CYC1 terminator)
- BBa_K4845007 (ADH1 terminator)
- BBa_K4845009 (temA)
These parts were added 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 genes on the X-3-backbone plasmid, improve the enzyme activity of glucoamylase, and further improve the ability of yeast to decompose starch.
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 the 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 a table and the form of a 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: Figure 2 : Visualized models of our plasmids designed (X-3-2temA)
Engineering Principle
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.
Characterization/Measurement
A. DNA sequencing of X-3-temA plasmid
According to the sequencing diagram shown, our X-3-temA and X-3-temA-2 plasmids were constructed successfully (Figure 3 and 4).
Figure 3: DNA sequencing result of X-3-temA plasmid
Figure 4: DNA sequencing result of X-3-temA-2 plasmid
B. Transformation testing of temA-containing plasmids through PCR and Gel electrophoresis
The figure 5 shown in figure A leads to the length of temA genes fragment of X-3 plasmid. We have successfully constructed those plasmids and we are able to confirm that we have successfully transformed our constructed temA-containing plasmid into 1974 yeast cells.
Figure 5: Results of PCR of plasmids extracted from temA-genes-containing 1974 yeast cell
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 3656
Illegal NotI site found at 4104 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 3877
Illegal XhoI site found at 6026
Illegal XhoI site found at 8750 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 6263
Illegal AgeI site found at 6697
Illegal AgeI site found at 8987 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 2651
Illegal BsaI.rc site found at 132
Illegal BsaI.rc site found at 4399
Illegal SapI site found at 284
Illegal SapI site found at 4213
Illegal SapI.rc site found at 1629
Illegal SapI.rc site found at 4636
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