Description
Biobrick is the essential for the synthetic Biology research, and it’s also a fundamental skill for the iGEM projects. At Kang Chiao international High School in Taipei, Taiwan, the school acknowledges how vital it is for high shool students to learn the synthetic biology research skills. The school has set up the syn/bio/res lab every Thursday from P5-8, and has started in September, 2021. Our team has learned the whole process of cloning concept and all of the techniques related to it, including PCR, designing restricted enzymes flanked on the forward and reverse primers, NEB double digestion, T4 ligation step, bacterial transformation on the selected plates from Sep,2021 to Jan, 2022. The class was separated into 3 groups and presented the project they were interested in and voted to come out the final project in Feb, 2022.
The rest of the 2nd semester from Feb to June of 2002, our team has worked on our parts. When constrctuing 4 different biobricks, our team used BY4741 strain’s genomic DNA to amplify the SNF1 entire gene. To manipulate SNF1 induction, the team will clone SNF1 entire gene into pGal1,10 promoter plasmid, galactose will be used to induce SNF1 gene drivn by pGal1,10 promoter. The pGal1,10-SNF1 plasmid will transform into wild-type BY4741 yeast strain as an overexpression SNF1 yeast strain in the presence of galactose; the pGal1,10-eGFP (green fluorescent protein)in BY4741 yeast strain is used as a control since no SNF1 protein will express in the presence of galactose. pGal1,10-snf1Δ2-306, N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation on Thr210 activates the catalytic activity of SNF1.
pGal1,10-snf1Δ381-633, C-terminus truncated from amino acid381 to 633 deleting autoinhibitory domain and SIP-interacting domain (SIR), losing the interaction with downstream of proteins. Those truncated SNF1 proteins, snf1Δ2-306 and snf1Δ381-633 are called dominant negative proteins, which might interference with normal SNF1 protein’s function. The purpose of having these two dominant negative proteins is to determine which domain causes more severe defective phenotypes. Our team created 4 basic parts, and 4 composite parts in this project. We thank the staff of the Taiwan Yeast Bioresource Center at the First Core Labs, National Taiwan University College of Medicine, For the mutation of SNF1, Δsnf1 in BY4741 yeast strain sharing.
In August, our team had 2 weeks summer internship to work on iGEM competition.
| Name |
Description |
Type |
Length |
| BBa_K4180000
|
SNF1in yeast is homology of AMPKα in human, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consumed(1) |
Coding |
1902bp |
| BBa_K4180001 |
Galactose promoter can be induced in the presence of galactose and suppressed in the presence of glucose(2) |
pGal1,10 promoter |
665bp |
| BBa_K4180002 |
N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation takes place on Thr210 to activate the catalytic activity of SNF1(3) |
Coding |
985bp |
| BBa_K4180003 |
C-terminus truncated from amino acid 381 to 633 deleting autoinhibitory domain and SIP-interacting domain (SIR) in the SNF1 protein(3) |
Coding |
1140bp |
| BBa_K4180004 |
(enhanced green fluorescent protein) is used as control
The original GFP from the jellyfish Aequorea victoria was not bright enough to show the gene expression or the protein localization of several different organisms. Yang et al lab did several mutations on the GFP DNA sequence to enhance the brightness of GFP to make the detection much more sensitive(4) |
Coding |
717bp |
| Name |
Description |
Length |
Diagram |
| BBa_K4180005 |
Galactose promoter can be induced in the presence of galactose and express SNF1(FL) ; SNF1 in yeast is homology of AMPKα in human, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consumed (1,4) |
2566bp |
 |
| BBa_K4180006 |
Galactose promoter can be induced in the presence of galactose to drive the expression of downstream coding gene, snf1Δ2-306aa (2,3)
|
1648bp |
 |
| BBa_K4180007 |
Galactose promoter can be induced in the presence of galactose and suppressed in the presence of glucose (2,3) |
1804bp |
 |
| BBa_K4180008 |
Galactose promoter can be induced in the presence of galactose and suppressed in the presence of glucose (2,4)
|
1381bp |
 |
