Why did we use yeast organisms?
Before making the stress testing kit, our team wanted to study the AMPK gene which is present in the human cell. However, given the limitations of our high school lab as well as safety concerns of using human cells in a P1 lab at high school’s experiment setting, we chose to use yeast as a model organism to do the experiment. The reasons that we used yeast to do the experiment are that: firstly, there are 25%-30% conserved genes between human and yeast cells, including AMPK in humans as a metabolism sensor, which has homology to SNF1 in yeast [1]. Students in the future can also create genetic modifications from yeast genes related to some human diseases; secondly, yeast is easy to manipulate and can also be stored in a -80 °C freezer (which we have available) for a long time; 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.
How did we decide to manipulate SNF1 only instead of other submits in the SNF1 family?
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 α subunit, β subunits, Sip1, Sip2 and Gal83 , and SNF1γ subunit, Snf4 [1]. Those different SNF1 subunits have functions related to metabolism, however, there are too many subunits (proteins) that we can’t study at the same time. The team can’t clone 3 different genes from SNF1α, β, γ into one plasmid, which will not be the proper 3D protein structures for any of them to have biological functions.
The act of transferring 3 plasmids into the yeast was too difficult, as the selection of yeast can be limited, as well the yeast having resistance to some antibiotics on the selection plates. Our team decided to only 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 consumed [2].
When our team did cloning steps with several different gene fragments, what problems did we encounter and what we did to solve them?
1. No band
When we ran the sample on the gel, there was no band. We realised that since the double enzyme digestions were incomplete, the DNA fragments were lost resulting in nothing being shown on the gel. Therefore, the team increased the plasmid concentration to start up, and also ordered KpnI and XmaI enzymes using the same buffer for digestion so we didn’t need to do sequential enzyme digestion anymore.
2. No sequence samples
When we sent out sequencing for our cloning samples to the MB mission Biotech company , we had a hard time getting those samples’ sequences done. Upon investigation the team found out that it was because we kept our plasmids in the TE buffer. The TE buffer is a Tris-EDTA, and EDTA binds with the protein/enzymes and inhibits the enzyme’s functions such as the enzyme for the gene sequence, causing the lack of results. Therefore, the TE buffer was later replaced by the ddH20(double distilled water) and the sequence was successfully seen.
How did our team know if our BioBricks (composite parts) really work and were really induced by galactose?
The 4 coding regions are downstream of the pGal1, 10 (BBa_K4180001) to generate 4 composite parts (BBa_K4180005, BBa_K4180006 , BBa_K4180007, and BBa_K4180008) which could be induced in the presence of galactose. After cloning those different basic parts using XmaI and KpnI double digestion to replace SPT5 gene on the plasmid to generate 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) as a control.
We also sent out sequences to the MB (Mission Biotech) company to confirm our cloning parts were correct. After creating those composite parts, our team also did a galactose induction time course to prove the pGal promoter system could be induced in the presence of 2% YP-galactose to check the induction of the coding regions on the composite parts via RT-qPCR technique. In the presence of galactose, the control of pGal1, 10-eGFP-SBP (BBa_K4180008) showed the maximum induction at least 20-fold eGFP mRNA induction at 41hr to indicate the pGal promoter system works properly in the presence of galactose.
Why did our team’s BY4741 transformed with pGal1, 10-eGFP-SBP (BBa_K4180008) show at least 20-fold induction in the presence of galactose, but not in others, pGal1, 10-SNF1-SBP (BBa_K4180005), pGal1, 10-snf1 Δ2-306-SBP (BBa_K4180006) , pGal1, 10-snf1Δ381-633 -SBP (BBa_K4180007) transformed into BY4741?
Firstly, it’s possible that BBa_K4180005, BBa_K4180006, and BBa_K4180007 coding regions were induced at the time courses but we didn’t collect samples in the presence of galactose, as our team might have missed the induction time course. Secondly, it’s possible that BBa_K4180005, BBa_K4180006, and BBa_K4180007 coding regions might have interfered with the endogenous SNF1 induction in the presence of galactose to cause the downstream genes of pGal promoter to be suppressed, or not overexpressed since some genes have negative feedback mechanisms. Finally, our team only has one chance to do galactose induction to check the genes’ mRNA levels via RT-qPCR once, it would be more promising to repeat the experiment at least 3 times. To solve these problems, we will work on collecting samples at different time courses in the presence of galactose, and repeat this at least 3 times to get consistent data.
Why did we use G418 on yeast SC-medium plates and YP-medium? What can be used as a control since in the presence of G418, BY4741 yeast wild-type strain can’t grow?
When doing transformation of different composite sites into BY4741, G418, a chemical reagent to suppress protein synthesis, was added into the selection plates. Only BY4741 with those composite sites having KanMX gene, which can grow on G418 toxicity on the selection plates. The pGal-eGFP (green fluorescent protein)in BY4741 yeast strain was cloned and used as a control since eGFP had no biological function in the yeast.
How did we add the supplements to the test plates
In order to assess the efficacy of the supplements on our test plates we needed to dissolve them into a solution to be added to the plates. We initially tried adding the supplements to water, and found they did not dissolve completely, which would result in an uneven concentration being applied to the plate sections. After enquiring with Hi-Q they supplied us with a new sample that was pure brown algae, and did not contain other materials added to the supplement, this sample dissolved successfully and we could use it on the test plates. The green tea supplement also did not dissolve with ethanol or low concentration acid solution, the final decision was to use lower concentrations as we realised we were beyond the saturation point.
1. Hedbacker, K, and Carlson, M. “SNF1/AMPK pathways in yeast.” Frontiers in bioscience : a journal and virtual library vol. 13 2408-20. 1 Jan. 2008, doi:10.2741/2854
2. Herzig, S. and R. J. Shaw (2018). "AMPK: guardian of metabolism and mitochondrial homeostasis." Nat Rev Mol Cell Biol 19(2): 121-135.
