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 part BBa_K4845004 (GAP promoter), BBa_K4000002 (CYC1 terminator), BBa_K4845007 (ADH1 terminator), and 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 genes on the X-3-backbone plasmid, improve the enzyme activity of glucoamylase, and further enhance the ability of yeast to decompose starch.

In order to construct BBa_K4845017 (X-3-temA-2), we first amplified the GAP and TEF1 promoters, temA key genes, and CYC1 and ADH1 terminators through PCR (Table 1). Since we were inserting 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 would be inserted into the plasmid skeletons (X-3) later (Figure 1). The design of our target genes is displayed both in the form of a table and a visualized diagram.

   <img src="" alt="Figure 1" width="400">

Figure 1: Visualized blocks-assembly diagram showing our design of the temA DNA template.<p> <p>Upper one: GAP-temA-CYC1 gene of 2804 bp.<p> <p>Lower one: TEF1-temA-CYC1 gene of 2482 bp.<p> <p> After obtaining our target temA gene fragments, we inserted them into X-3 plasmid skeletons using the restriction endonuclease digestion and ligation method (Figure 2).

   <img src="" alt="Figure 2" width="400">

Figure 2: Visualized models of our designed plasmids (X-3-2temA).<p>

Engineering Principle

<p>

   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, while the α-1,6 glycosidic bond and α-1,3 glycosidic

bond were slowly hydrolyzed, resulting in the final product being 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).

   <img src="" alt="Figure 3" width="400">

Figure 3: DNA sequencing result of X-3-temA plasmid.<p> <img src="" alt="Figure 4" width="400"> <p>Figure 4: DNA sequencing result of X-3-temA-2 plasmid.<p> <p>B. Transformation testing of temA-containing plasmids through PCR and Gel electrophoresis

Figure 5 shows the length of temA gene fragments 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.

   <img src="" alt="Figure 5" width="400">

Figure 5: Results of PCR of plasmids extracted from temA-genes-containing 1974 yeast cells.<p> Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 3656
  Illegal NotI site found at 4104
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 3877
  Illegal XhoI site found at 6026
  Illegal XhoI site found at 8750
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 6263
  Illegal AgeI site found at 6697
  Illegal AgeI site found at 8987
  
• 1000
  INCOMPATIBLE 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