Troubleshooting
1. While making the agar plates, we tried to put the brown algae and green tea extract supplements onto the agar plate to test our product, but they could not dissolve completely. To solve this issue, we decided to autoclave the supplements since a higher temperature could increase the solubility of the supplements while the higher pressure could make the liquids remain liquid under high temperature. Adding chemical buffers that could dissolve the solvent, including ethanol, acidic and basic solution, are also beneficial in assisting the supplements to dissolve. These problems may also be encountered by other IGEM teams, so autoclaving materials and adding chemical buffers may be potential solutions to solve similar issues.
2. When we tried sending our cloning samples to the Mission Biotech company to confirm our sequence, They had a hard time confirming it. This is because we delivered our samples in the TE buffer, which contains EDTA inside. The EDTA compound in the buffer may bind with specific enzymes and proteins, inhibiting their function, thus hindering the sequence confirmation. Consequently, we decided to replace the TE buffer with dH2O to eliminate the effect of EDTA. It is important for future teams to pay attention to the substance when delivering the DNA sequences, and to avoid using TE buffers for delivery.
3. Our promoter is expressed in the presence of galactose, which drives the downstream genes to also be expressed. We do not know when our plasmid is expressed thus cannot assume all the phenotype mitigation on our plates to be caused by the overexpression of our target gene. Therefore, we conducted galactose induction with time course to collect samples of our plasmid in the presence of 0’, 30’, 1h, 17h, 24h, 41h 2%YP-galactose, which are then used to conduct RT-qPCR to identify the time when the target gene is significantly expressed. For experiments that are also working with SNF1 and its truncation forms, as well as other experiments that aim to test the expression of a promoter, the use of time course can be a good method to obtain specific time and results.
4. During the cloning process, we were not able to clone and conserve AMPK alpha, beta, and gamma into one single yeast plasmid, as the protein structure could not fold properly when AMPK alpha, beta, and gamma are consecutive downstream in a single plasmid. On the other hand, while there is an alternative to clone alpha, beta, and gamma in three different plasmids into the plasmid, the end result could be inconsistent. Therefore, we decided to use AMPK alpha only because it is upstream. Future IGEM teams should take note of the amount of genes to clone while paying attention to whether it would influence the folding of proteins, or whether it could yield valid and consistent results.
5. During double enzyme digestions on Kpn1 and Xma1 sites, we found out that the sample is lost. We suspect the possibilities of the inconsistent experiment execution that causes the sample loss, yet this phenomenon is observed throughout numerous double enzyme digestion trials. Therefore, we increased our plasmid concentration to make up for the deficit. For experiments related to double digestion, it is important for experimenters to be aware of whether the digestion is done correctly to prevent the loss of DNA information.
Information
SNF1, the homology of AMPK conserved in yeast, plays an important role in the response to cellular stress in yeast. By the introduction of SNF1, future team are allowed to conduct experiment simulating AMPK in the P1 lab. AMPK not only serve a main role in regulating metabolism but also in mitochondrial homeostasis. Furthermore, in our project, we designed to use carbon deprivation for heat shock tests. By shifting glucose, the main food source of yeast, to galactose, glucose starvation triggers the yeast to overexpresses the downstream genes. This knowledge enable future teams, who are studying and researching environmental stresses on yeast, a method for triggering specific activation of yeast’s function.
Data
RT-qPCR data
In our project, we engineered our genes downstream of a plasmid with pGal promoter, which activates in the presence of galactose. Therefore, we needed to find out when our downstream gene is activated in the presence of galactose, which is assumed to be the same as when the downstream gene expresses during the survival plate assays. We will know the change in the stress-induced-phenotype on yeast (whether it mitigates) will have a high probability to be caused by the downstream gene expression if stress-induced-phenotype is mitigated at the same time it was shown to be expressed in the presence of galactose.
To this mean, we conducted Galactose Induction with Time Course to find out the time course when the downstream gene is expressed the most. We would put respective downstream gene in 2% YP-galactose for 0’, 30’, 1h, 17h, 24h, 41h (chosen because of school schedule), and conduct RT-qPCR to find the number of folds for each time course, which is used to indicate when genes are expressed. The data are the following:
| Endogenous SNF1 in BY4741 (wild type, use SNF1-2 primers to do qPCR) |
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| BBa_K4180008 in BY4741 (use eGFP primers to do qPCR) |
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| BBa_K4180005 in BY4741 use SNF1-1 primers to do qPCR |
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| BBa_K4180006 in BY4741 use SNF1-2 primers to do qPCR |
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| BBa_K4180007in BY4741 use SNF1-1 primers to do qPCR |
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