Coding

Part:BBa_K4000001

Designed by: Ke ZHU   Group: iGEM21_Fujian_United   (2021-10-12)
Revision as of 10:26, 9 October 2023 by Ghx0311 (Talk | contribs)


GA


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Improvement by Team iGEM23_SubCat-China

BBa _ K4000001 (GA)

Group: SubCat-China iGEM 2023

Summary:

We constructed a new recombinant plasmid BBa _ K4845020( X-2-GA-2 ) based on the old part BBa _ K4000001 (GA)...

Construction Design and Engineering Principle

The problems we are going to solve are... [the rest of your text]...

Figure1: Overview of the methodology of our project design

Figure1: Overview of the methodology of our project design through synthetic biology (made in Canva.cn)

Characterization/Measurement

A)Method of Transparent Circle

According to the property of starch... [the rest of your text]...

Figure 2: Transparent circle experiment for the function testing

Figure 2: Transparent circle experiment for the function testing...

B)Enzyme Activity Detection in Starch Hydrolyzing Capacity

To verify the GA and temA activity... [the rest of your text]...

Figure 3: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974

Figure 3: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974...

Figure 4: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974

Figure 4: The enzyme activity of Yeast 1974-GA-temA and Wild Yeast 1974...

Figure 5: The comparison of the enzyme activity curves of Yeast 1974-GA-temA

Figure 5: The comparison of the enzyme activity curves of Yeast 1974-GA-temA...

C)Determination of alcohol production

To further and directly verify... [the rest of your text]...

Figure 6: Alcohol production of yeast 1974 and GA-temA-1974

Figure 6: Alcohol production of yeast 1974 and GA-temA-1974

Reference:

  1. [2] Xu Shuai, Yang Li, Tan Liping, et al. Enzymatic hydrolysis and application of sweet potato residue [ J ]. Journal of Qilu University of Technology. 2021, 35(03): 28-33. [3] Xin Wang, Bei Liao, Zhijun Li, et al. Reducing glucoamylase usage for commercial-scale ethanol production from starch using glucoamylase expressing Saccharomyces cerevisiae[J]. Bioresources and Bioprocessing, 2021, 8(1): 20. [4] Rosemary A. Cripwell, Lorenzo Favaro, Marinda Viljoen-Bloom, et al. Consolidated bioprocessing of raw starch to ethanol by Saccharomyces cerevisiae: Achievements and challenges[J]. Biotechnology Advances, 2020, 42: 107579. [5] LM de Moraes, S Astolfi-Filho, SG Oliver. Development of yeast strains for the efficient utilisation of starch: evaluation of constructs that express alpha-amylase and glucoamylase separately or as bifunctional fusion proteins[J]. Applied microbiology and biotechnology, 1995, 43(6): 1067-76.


Profile

Name: GA

Base Pairs: 1567bp

Origin: Saccharomycopsis fibuligera, synthesis

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.

Figure1. Principle diagram of GA..

Experimental approach

1.GA plasmids transformation

Figure 2. GA plasmids transformed Saccharomyces cerevisiae..

GA-expressing plasmids transformation and positive S. cerevisiae transformants selection using 50 μg/mL (Figure 2A) and 350 μg/mL (Figure 2B) hygromycin. Top left, pYES2-ctl. Top right, pYES2-TGC. Bottom left, pYES2-HGC. Bottom right, pYES2-IGC.


2. Colony PCR to identify the correct transpormed S. cerevisiae

Figure 3. Verification of the plasmids pYES2-ctl (A), pYES2-TGC (B), pYES2-HGC and pYES2-IGC (C) transformation via colony PCR in CEN.PK2-1C strain..


The colonies which grew on the high concentration hygromycin plates were subjected to colony PCR to verify the plasmids transformation again. From Fig. 3 we can see that positive bands implied the plasmids transformation successfully.


Proof of function

1. Glucoamylase activity assay

Figure 4. Glucoamylase activity assay regulated by different promoters..

S. cerevisiae strains harboring various plasmids glucoamylase activity determination. **Statistical significance between indicated strains by Student’s t test, p < 0.01. ns, not significant. Data represent the means of two independent colonies.

2. Fermentation test

Figure 5. Glucose concentration inside the cell during the GA-expressing S. cerevisiae strains corn starch fermentation process..

To verify the GA secretion capacity of the GA-expressing S. cerevisiae strains, we measured the glucose concentration inside the cell during the corn starch fermentation process. At the initial stage (0 h), when the GA was added during the “starch-to-glucose” process, higher contents of glucose were detected than the process without GA addition.


References

1. Görgens J F, Bressler D C, van Rensburg E. Engineering Saccharomyces cerevisiae for direct conversion of raw, uncooked or granular starch to ethanol[J]. Critical reviews in biotechnology, 2015, 35(3): 369-391.

2. Van Zyl W H, Bloom M, Viktor M J. Engineering yeasts for raw starch conversion[J]. Applied microbiology and biotechnology, 2012, 95(6): 1377-1388.

3. Maury J, Kannan S, Jensen N B, et al. Glucose-dependent promoters for dynamic regulation of metabolic pathways[J]. Frontiers in bioengineering and biotechnology, 2018, 6: 63.

4. Weber E, Engler C, Gruetzner R, et al. A modular cloning system for standardized assembly of multigene constructs[J]. PloS one, 2011, 6(2): e16765.

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 131
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 92
    Illegal AgeI site found at 1463
  • 1000
    COMPATIBLE WITH RFC[1000]


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