1. The team conducts the PCR technique to amplify the specific genes products and successfully observes the bands on the electrophoresis gel
Before cloning the genes we are interested in, the team conducts the PCR technique to amplify the specific genes products. The PCR products are shown on the figure above.
2. The team used the sample collected from the bacteria colonies to conduct PCR. The figures above show the PCR products demonstrating that the genes are cloned successfully into the plasmid
3. Mission Biotech (MB) confirms that our cloned sequences are correct
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 sent out sequences to the MB (Mission Biotech) company to confirm our cloning parts were correct.
4. Galactose induction time course proves pGal promoter system works
After creating the 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.
BBa_K4180008 in BY4741 (use eGFP primers to do qPCR)
5. Cell viability plates showing that the heat shock stress and carbon deprivation stress worked on the plates
Fig 8: Brown algae extract itself didn’t alleviate any stresses in the environment.
In the control plate(Fig8A), wild type BY4741 and ΔSNF1 strains showed normal phenotypes with similar growth in 2% glucose-SC medium; however, in the presence of 2% galactose-SC, the carbon deprivation stress conditions, wild type BY4741 and ΔSNF1 strains showed severe growth defective phenotypes (Fig8B) compared to the strains under normal condition (FIg8A). In addition, ΔSNF1 strain had more drastic growth defective phenotypes compare to BY4741(Fig8B) in the presence of 2% galactoe-SC, indicating that our team’s carbon deprivation stress with 2% galactose-SC medium plate did induce defective phenotypes related to the SNF1 pathways that the ΔSNF1 strain, with more environment sensitive, as stated above, would experience more drastic defective phenotypes.
To further test whether the heat shock worked on the plate, the control plate(Fig8A) had a more significant growth compared to the 2% glucose-SC with 37 degree celsius heat shock(Fig8D). The same difference can be seen in both Fig8B and Fig8E as well as Fig8C and Fig8F, showing that the heat shock had acted like a stress to the performance of the growth of yeast.
At fig 9, our team started with single stress, heat shock at 37°C in 2%glucose-SC-medium (9C), and pGal-eGFP in BY4741 and pGal-SNF1(FL) in BY4741 showed slightly growth defective phenotypes (9C) compared to the normal growth temperature (30°C) in 2%glucose-SC-medium (9A); pGal-Δ2-306aa in BY4741 strain showed dramatic growth defective phenotype at 37°C (9C) compared to it at 30°C (9A). Through this data, we can confirm that our heat shock stress did have an impact on the yeast growth. In the presence of 2% galactose as the carbon deprivation stress condition, all strains showed drastic severe growth defective phenotypes (10B) compared to the ones in the normal condition (10A). Like the heat shock stress, the drastic difference in growth between the plates that have and don’t have carbon deprivation is significant, showing that our carbon deprivation stress works in disrupting growth for the yeast.
To determine whether our overexpression of SNF1 related genes(snf1Δ2-306aa, snf1Δ381-633aa, and SNF1 full-length) downstream of pGal promoter combined with brown algae extract showed synergistic effects on the mitigation on the growth defective phenotypes induced by environmental stresses, our team performed an experiment, shown on Fig9 and Fig10, by manipulating either heat shock stress, carbon deprivation stress, or both on yeast growth medium plates to perform cell viability assay.
For testing whether the supplements help mitigate stress for the yeast strains that had overexpressed SNF1 related genes(snf1Δ2-306aa, snf1Δ381-633aa, and SNF1 full-length), our data from figure 9 does prove that. When comparing pGal-snf1Δ2-306aa in BY4741 for 9A and 9B, the survival plates show that 9B, which has 100ug/ul brown algae extract does help mitigate stress as there’s a dramatic growth of yeast shown on the plate. The same can be seen in figure 9C and 9D. When combined with the 100 ug/ul brown algae extract, the pGal-snf1Δ2-306aa in BY4741 strain again showed significant growth for the plate that had the supplement compared to the plate that did not have the supplement. The data comparisons from the two plates, shows that the brown algae nutrient did help mitigate the stress that the yeast receives to some degree.
6. Brown Algae extract combined with overexpression of pGal-SNF1Δ2-306aa in BY4741 shows a result
Fig9 : In the presence of brown algae extract along with overexpression of pGal-SNF1Δ2-306aa in BY4741 reduced growth defective phenotype induced by heat shock . The pGal-SNF1Δ2-306aa growth defective phenotype at normal conditions was also reduced.
Fig10 : Brown algae extracts combined with overexpression of our composite parts BBa_K4180005, BBa_K4180006, BBa_K4180007, and BBa_K4180008 under all circumstances of heat shock single stress, carbon deprivation single stress, and double stress on cell viability growth medium plates did not show any significant synergistic effect in mitigating the growth defective phenotypes induced.