KCIS_Xiugang_Taipei

Goal

   Our team’s final mission is to to design a stress testing kit, which can detect which local nutrients’ supplements, such as brown algae extract or green tea extract, can alleviate the stresses in the environment, and combing with the overexpression of a metabolistic sensor gene can show synergistic mitigation stresses in the environment via cell viability assay [15].

Choosing Target Gene

   With the topic of our project targeting human metabolism to reduce obesity, our primary target gene of choice is AMPK, a master regulator of energy that is directly related to metabolistic functions within the human body. Upon induction by upstream pathways, AMPK will boost metabolism, triggering fatty acid catabolism while suppressing anabolism for more ATP. Research showed that stresses, such as lipotoxicity and intracellular lipid reactive oxygen species (ROS), are harmful to DNA and protein structures [6] [12] and also triggers the expression of AMPK. Misregulation of AMPK expression in type II diabetic patients, at the category of kidney-related injury, caused renal lipotoxicity [8][9][10], which indicated that AMPK can be induced by stresses to cope with the environmental changes.

   For the purpose of our project design at a high school level lab and considerations regarding the accessibility of human cells from real life donors, we selected the gene SNF1 as our main target gene since it is found to be homologous to AMPK genes and its functions are highly conserved in eukaryotes. In the null mutation of SNF1 in yeast, Δsnf1, is sensitive to environmental stresses, such as glucose deprivation, heat shock, and alkaline PH, and ROS stress and metal stress [3][1]. These research results further prove SNF1’s gene similarity and homology to AMPK genes, making it the optimal candidate for further cloning processes and experimental designs that will fully model the AMPK gene functions and characteristics.

   Regarding the transformation of genes for their functional expression, we chose to use yeast as a model organism. The main reasons are that firstly since there are 25%-30% conserved genes between humans and yeast, including AMPK in humans as metabolism sensor homology in yeast [3], students in the future can also create genetic modifications from yeast genes related to some of human’s diseases. Secondly, yeast organisms are easy to manipulate and can also be stored in a -80 degree freezer for a long time, which allows a more sufficient time span for conducting experiments and allows more long-term studies to be conducted on the edited gene products. Thirdly, yeast genes rarely have introns so it’s easier for high school students to create BioBricks using yeast genomic DNA directly instead of cDNA. And finally, it is possible to use these in the setting of our lab which fits the P1 experiment setting.

   With more detailed investigations into our target gene, the AMPK family in human cells is composed of three components AMPKα, AMPKβ, and AMPKγ, which are also conserved in yeast cells, however, in yeast, SNF1 encodes the catalytic alpha subunit, β subunits having Sip1, Sip2 and Gal83 , and SNF1γ subunit, Snf4 [3]. The different SNF1 subunits have functions related to metabolism, however, there are too many subunits (proteins) that we can’t study at the same time. To reach our team’s final goal, we decided to manipulate SNF1, 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 consume [4].

   Within our SNF1 gene cloning, we also included 2 sets of truncation in order to observe which parts of the gene play more of a significant role in its metabolic functions. There are two truncations: N truncate and C truncate. The N terminal is responsible for the catalytic activities of SNF1. Therefore, the N truncate removes its catalytic activity. C terminal is responsible for interaction with other proteins, so C truncate removes its ability to interact with other proteins.

Biobrick

   Our team had to overexpress the SNF1 protein so we cloned SNF1 full length downstream of a pGal promoter plasmid, using the following protocols.

1. Yeast genomic DNA extraction from BY4741 genomic DNA
2. PCR to get more genomic DNA samples, run PCR product on agarose gel
　　[protocol: gel electrophoresis]
3. NEB double enzyme digestion of the genomic DNA sample to collect SNF1 from the endogenous genomic DNA
4. NEB double enzyme digestion on [pGal1, 10-SPT5-SBP(Streptavidin Binding Protein) plasmid, which was sponsored by Dr. Tien-Hsien Chang, at Genomics Research Center, Academia Sinica in Taipei, Taiwan.
5. T4 ligation to ligate the SNF1 sample onto the cutting site
6. Sent sequence to Mission Biotech for confirmation
7. Transform the plasmid in E. Coli to create colonies
8. Extract the plasmid from E. Coli colonies and transform it into BY4741

   The pGal-eGFP (green fluorescent protein) in BY4741 yeast strain was cloned and used as a control to validate the functionality of our pGal promoter system since eGFP had no biological function in the yeast organism using a similar protocol as above.

   In addition to the full-length SNF1 and the control eGFP, we would like to determine which domain of the SNF1 gene causes more severe defective phenotypes. Therefore, we truncated the 2 domains of the SNF1 full-length gene, forming SNF1Δ2-306 and SNF1Δ381-633 which both might interfere with the endogenous SNF1 protein’s function. SNF1Δ2-306 or SNF1ΔN, the N-terminus truncation, has its amino acid 2 to the 306, the kinase domain of SNF1, where the phosphorylation takes place on Thr210 to activate the catalytic activity of SNF1 tuncated [11]. SNF1Δ381-633 or SNF1ΔC, the C-terminus truncation, has its amino acid 381 to 633, the autoinhibitory domain and SIP-interacting domain (SIR) deleted [3]. The truncated SNF1 proteins, called dominant negative proteins, are cloned as downstream of a pGal promoter driven plasmid individually using a similar protocol listed above.

   With the 5 basic parts (SNF1, SNF1ΔN, SNF1ΔC, eGFP; BBa_K4180000, BBa_K4180002, BBa_K4180003, BBa_K4180004) and the inducible promoter (pGal; BBa_K4180001) that is induced in the presence of 2% galactose, our team has created 4 composite parts for this project: pGal-SNF1, pGal-SNF1ΔN,p pGal-SNF1ΔC, and pGal-eGFP; BBa_K4180005,BBa_K4180006,BBa_K4180007,BBa_K4180008 respectively.

   To test the viability of our product we need to not only use BBa_K4180008, we also use wild type BY4741 (without any plasmid transforms or mutations) and ΔSNF1 in BY4741 for phenotype comparison. We thank the staff of the Taiwan Yeast Bioresource Center at the First Core Labs, National Taiwan University College of Medicine, for sharing the BY4741 yeast strain with SNF1 mutation, the BY4741 ΔSNF1.

    

Test

   After creating the four composite parts, the team did galactose induction with time course to prove those composite parts could be induced in the presence of 2% YP-galactose to check the induction of the coding regions on the composite parts via RNA extraction and RT-qPCR technique [Protocol: RT-qPCR]. BY4741 as wild-type and Δsnf1 as a mutant in the genome, without composite parts, were used along with BY4741 transformed pGal1, 10-eGFP-SBP (BBa_K4180008) as controls shown in the figure below.

Figure 1: The team resuspended the collected yeast from the 2% glucose-YP + G418 medium by using the ddH2O until it reaches OD~0.4 and transferred them into the 2% galactose-YP medium to collect the time course samples(set up at 0 minutes, 30 minutes, 1 hour, 17 hours, 24 hours and 41 hours) for RT-qPCR to detect the downstream genes of mRNA.

Environmental stresses design

  

To induce the stresses in the environment, the team used the heat shock stress at 37 degree celsius while adding galactose instead of glucose to cause carbon deprivation stress that can be manipulated on the yeast growth medium plates. By creating double stresses, it mimics the condition of the human body. In the presence of galactose, it not only causes carbon deprivation stress, but also induces the downstream genes of the pGal promoter.
   The cloned composite sites pGal-SNF1, pGal-snf1Δ2-306, pGal-snf1Δ381-633 plasmids will be transformed into yeast wild-type BY4741, respectively. Δsnf1 in BY4741, along with 3 different SNF1 cloned plasmids transformed into BY4741 to do a cell viability assay [15] on the double stresses medium to determine which strain grow better or worse by comparing the pGal-eGFP transformed in BY4741 and original wild-type, BY4741.

   The experimental designs are below.

Figure 2: The cell viability experiment is set up with 2 different stresses, such as carbon deprivation by adding galactose instead of glucose along with heat shock stress by putting the plates at 37 degree v.s 30 degree (those stresses in the environment can change).

  

BY4741 is a wild-type strain without any effects in the presence of the stresses environment; Δsnf1 in BY4741 yeast strain and Δhsp70 in BY4741 yeast strain are used as controls to show carbon deprivation stress and heat shock stress work properly on the cell viability plates by showing defective phenotypes. If the local nutrient supplements mitigate stresses in the environment, we expect to see less defective phenotype on those mutant strains, such as Δhsp70 and Δsnf1 compared to the cell viability plates without nutrient supplements.

Figure 3: The combination of overexpression of our team’s cloning genes driven by pGal promoter with the brown algae extract shows the synergistic effects on either single stress, such as carbon deprivation by adding galactose instead of glucose or heat shock stress by putting the plates at 37 degree v.s 30 degree or both on the cell viability plates
  

There are 4 different composite parts, pGal1, 10-SNF1-SBP (BBa_K4180005), pGal1, 10-snf1 Δ2-306-SBP (BBa_K4180006) , pGal1, 10-snf1Δ381-633 -SBP (BBa_K4180007), and pGal1, 10-eGFP-SBP (BBa_K4180008) and those composite parts will transform into BY4741 yeast strain [Protocol: yeast transformation]. pGal1, 10-eGFP-SBP (BBa_K4180008) in BY4741 is used as a control without any effects in the presence of the stress environment, Δsnf1 in BY4741 yeast strain and Δhsp70 in BY4741 yeast strain are used as controls to show carbon deprivation stress and heat shock stress work properly by showing defective phenotypes on cell viability plates. If the combinations of overexpression SNF1 with local nutrient supplements, such as brown algae extract and green tea extract, respective, show any synergistic effect on the mitigation of stresses in the environment, we will expect to see drastic alleviation of the defective phenotype on those mutant strains, such as Δhsp70 and Δsnf1 compared to the cell viability plates without nutrient supplements.