Difference between revisions of "Part:BBa K4000004"

Latest revision as of 07:33, 20 October 2021

ICL1p

Profile

Name: ICL1p

Base Pairs: 401bp

Origin: Saccharomyces cerevisiae, genome

Properties: Inducible promoter regulated by glucose

Usage and Biology

The ICL1 promoter displays high levels of induced expression; however, ICL1 repression was incomplete in the presence of glucose.

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 1. Schematic map of Glucoamylase expression plasmids. ICL1p is promoter..

Experimental approach

1. Fragments PCR products Electrophoresis

Figure 2. 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.

2. Colony PCR to identify the correct plasmids

Figure 3. 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 4. GA plasmids transformed Saccharomyces cerevisiae and verification..

GA-expressing plasmids transformation and positive S. cerevisiae transformants selection using 50 μg/mL (Figure 4A,D) and 350 μg/mL (Figure 4B,E). The colonies which grew on the high concentration hygromycin plates were subjected to colony PCR to verify the plasmids transformation again. From Fig. 4 we can see that positive bands implied the plasmids transformation successfully.

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