Part:BBa_K4000007

ICL1p+GA+CYC1t

Usage and Biology

Alcohol (ethanol) is an important chemical in the fields of food, medicine and bio-fuel. In particular, since the outbreak of COVID-19, the demand for alcohol for disinfection has skyrocketed [1]. Liquefaction enzyme expression in Saccharomyces cerevisiae has been reported. Bacillus amyloliquefaciens, Bacillus stearothermophilus, Bacillus Licheniformis, Bacillus subtilis Subtillis, Streptococcus Bovis, Debaryomyces Occidentali, Saccharomycopsis fibuligera and barley are common sources of α -amylase [2-3]. Because that the yeast must secrete enough amylase to support its growth at a higher initial starch concentration, and only growth can secrete enough amylase. This paradox leads to a prolonged yeast fermentation cycle, which cannot meet the requirements of industry.

Based on the above studies [4-5], we considered codon optimized Glucoamylase gene (GA) derived from S. fibuligera. After elements optimization, the saccharification enzyme activity of culture supernatant of the strain obtained and the alcohol production capacity of fermented corn starch subenzyme were tested in order to construct the proper strain.

Figure1. Principle diagram of GA..

Construct design

GA (Glucoamylase) is known as the type of enzyme that can easily break down starches into glucose, which afterwards becomes usable and absorbable. we use the constitutive ICL1 promoter to regulate GA.

Figure 2. Schematic map of Glucoamylase expression plasmids. pYES2-ctl is control plasmid..

The profiles of every basic part are as follows:

BBa_K4000002

Usage and Biology

This is a common transcriptional terminator. Placed after a gene, it completing the transcription process and impacting mRNA half-life. This terminator can be used for in vivo systems,and can be used for modulating gene expression in yeast.

BBa_K40000001

Origin: Saccharomycopsis fibuligera, synthetic

Properties: An enzyme that can easily break down starches into glucose

Usage and Biology

Glucoamylase is an enzyme that can be obtained from the yeast or fungi in the Aspergillus genus such as Aspergillus niger. The enzyme decomposes starch molecules in the human body into the useful energy compound of glucose. This is accomplished by removing the alpha-1 and 4-glycosidic linkages from the non-reducing end of the starch molecule. These molecules are more commonly referred to as polysaccharides and are frequently either amylase- or amylopectin-based.

Experimental approach

1. Fragments PCR products Electrophoresis

Figure 3. Gel electrophoresis of amplified fragments..

The basic parts of the plasmids such as the pYES2 backbone, GA coding sequence, ICL1 promoters, and terminators were all amplified successfully firstly. Then the multiplex GA expression cassettes were assembled using the overlap PCR method.

2. Colony PCR to identify the correct plasmids

Figure 4. Gel electrophoresis of colony PCR..

We chose 24 colonies to verify whether the plasmids were correct or not using the colony PCR, the positive rate for the plasmids pYES2-ICL1 were 17/24. Two or three positive colonies were sequenced to verify further.

3. GA plasmids transformation and identification

Figure 5. GA plasmids transformed Saccharomyces cerevisiae and verification..

GA-expressing plasmids transformation and positive S. cerevisiae transformants selection using 50 μg/mL (Figure 5A,D) and 350 μg/mL (Figure 5B,E).

The colonies which grew on the high concentration hygromycin plates were subjected to colony PCR to verify the plasmids transformation again. From Fig. 5 we can see that positive bands implied the plasmids transformation successfully.

References

5. Pollak B, Cerda A, Delmans M, et al. Loop assembly: a simple and open system for recursive fabrication of DNA circuits[J]. New Phytologist, 2019, 222(1): 628-640.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 532
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 493
  Illegal AgeI site found at 1864
  
• 1000
  COMPATIBLE WITH RFC[1000]