Experimental Results for Galactose Induction with Timecourse
pGal1, 10-SPT5-Streptavidin Binding Protein(SBP) plasmid, containing 2u origin of replication (ORI) for yeast to start DNA replication, and ORI for bacteria DNA replication, was sponsored by Dr.Tien-Hsien Chang, at Genomics Research Center, Academia Sinica, in Taipei Taiwan. This plasmid can be transformed into bacteria and yeast, which was beneficial for our team to finish making biobricks in bacteria and perform the functional assay in yeast. Our team made 5 different basic parts (one promoter and 4 coding regions) and 4 different composite parts by inserting those 4 basic parts (BBa_K4180000, BBa_K4180002, BBa_K4180003, and BBa_K4180004) to replace the original SPT5 gene. 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 cloned 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 the sequence to confirm our composite parts are correct. Furthermore, the team also did the galactose induction time course to prove those composite parts were induced in the presence of 2% YP-galactose to check the induction of the coding regions on the composite parts via RT-qPCR. BY4741 and ΔSNF1, without composite parts, were used as controls.
BY4741 (Wild type)use SNF1-2 primers
Fig 1: In the presence of 2% YP-galactose, the native SNF1 was also normally induced since the food source has changed to a carbon-deprivation source. In the time course experimental data, the endogenous SNF1 was induced over 3 fold-induction in the presence of 2%YP-galactose for 17hr, close to 7-fold induction in 24hr, and 5-fold induction in 41hr.
𝚫SNF1 in BY4741(k/out)use SNF1-2 primers
Fig2 : 𝚫SNF1 in BY4741 has partial SNF1 coding region deleted in the genome so our team used it as a negative control. In the presence of 2%YP-galactose, the induction of SNF1 was not manipulated until 41 hr later to show around 2.5-fold induction, which showed drastic reduction of SNF1 compared to the one BY4741 wild type.
BBa_K4180008 in BY4741 (use eGFP primers to do qPCR)
Fig 3: BBa_K4180008 was used as a control for BBa_K4180005, BBa_K4180006, BBa_K4180007 composite parts transformed into BY4741 yeast strain, respectively. The purpose of doing a timecourse in the presence of galactose was to determine whether our composite part could be induced by the galactose. In the presence of 2% YP-galactose for 17hr, eGFP was induced over 10-fold induction, even reaching over 20-fold induction in the presence of 2% YP-galactose for 41hr, which indicated our team’s composite parts could be manipulated to dramatically induce the coding region downstream of Gal1,10 promoter in the presence of galactose.
BBa_K4180006 in BY4741 use SNF1-2 primers to do qPCR
Fig 4: BBa_K4180006 composite site was SNF1 N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation took place on Thr210 to activate the catalytic activity of SNF1, cloned downstream of the Gal1, 10 promoter. BBa_K4180006 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced less than 2-fold at 24, and 41 hr, which didn’t show if the BBa_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in BBa_K4180008 control, since BBa_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180006 composite site was suppressed in the presence of galactose.
BBa_K4180005 in BY4741 use SNF1-1 primers to do qPCR.
Fig 5: BBa_K4180005 composite site was SNF1 full length coding region cloned downstream of the Gal1, 10 promoter. BBa_K4180005 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 3-fold at 17, and 24 hr, and 5-fold at 41 hr, which didn’t show if the BBa_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in BBa_K4180008 control, since BBa_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180005 composite site was suppressed in the presence of galactose at 24hr.
BBa_K4180007in BY4741 use SNF1-1 primers to do qPCR
Fig 6: BBa_K4180007 composite site was SNF1 C-terminus truncated from amino acid381 to 633 deleting autoinhibitory domain and SIP-interacting domain (SIR) cloned downstream of the Gal1, 10 promoter. BBa_K4180007 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 2-fold at 17, and 24 hr, and 3.5-fold at 41 hr,which didn’t show if the BBa_K4180007 composite site was manipulated in the presence of galactose, compared to the induction in BBa_K4180008 control, since BBa_K4180007 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa_K4180007 composite site was suppressed in the presence of galactose at those timecourses.
Conclusion for Galactose-Induction with Time Course of the pGal1, 10 Promoter Driven Downstream of Coding Regions
BBa_K4180008 was used as a control for BBa_K4180005, BBa_K4180006, BBa_K4180007 composite parts transformed into BY4741 yeast strain, respectively. The purpose of doing a time course in the presence of galactose was to determine whether our composite part could be induced by the galactose. In the presence of galactose, the control GFP maximum induction showed at least 20-fold induction at 41hr, which clearly showed the BBa_K4180001 basic part could be manipulated easily by the presence of galactose. However, BBa_K4180006 composite site was SNF1 N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation took place on Thr210 to activate the catalytic activity of SNF1, cloned downstream of the Gal1, 10 promoter; BBa_K4180005 composite site was SNF1 full-length coding region cloned downstream of the Gal1, 10 promoter; BBa_K4180007 composite site was SNF1 C-terminus truncated from amino acid381 to 633 deleting the autoinhibitory domain and SIP-interacting domain (SIR) cloned downstream of the Gal1, 10 promoter, three composite sites didn’t show strong manipulation in the presence of galactose. There are several possibilities as to what might have occurred; first, it’s possible that BBa_K4180005, BBa_K4180006, and BBa_K4180007 coding regions were induced before 17 hr of the presence of galactose, which our team didn’t observe. The second possibility was that it’s possible that BBa_K4180005, BBa_K4180006, and BBa_K4180007 coding regions might have interfered with the endogenous SNF1induction in the presence of galactose to cause the SNF1 induction’s suppression.
Results for Yeast Survival Plates
The null mutation of SNF1 in yeast, Δsnf1 was sensitive to environmental stresses such as glucose deprivation, heat shock, and alkaline PH, and ROS stress and metal stress et al., (Hedbacker and Carlson 2008, Casamayor, Serrano et al. 2012) so due to the time sensitivity to not be able to get Δhsp70 as heat shock control on time, our team decided to use Δsnf1 in BY4741 as glucose deprivation and heat shock stresses as a control strain.
Stress induced in the environment by incubating plates at 37°C (heat shock stress) and by replacing glucose in the supplements with galactose (Carbon deprivation), not only induced stress on yeast, but also induced the downstream genes we cloned on the pGal driven plasmid transformed into BY4741. By manipulating heat shock and carbon deprivation respectively and simultaneously on yeast growth medium plates, single stress and double stresses was induced, which mimics the conditions the human body experiences.
The cloned composite sites, BBa_K4180005, BBa_K4180006, and BBa_K4180007, transformed in BY4741 yeast, along with ΔSNF1 in BY4741 were used to demonstrate the cell viability assay (15) on growth medium plates induced with either carbon deprivation, heat shock, or both. The results of these strains was compared with BBa_K4180008 and wild type BY4741.
Fig7: Brown algae extract itself didn’t alleviate heat shock stress nor improve growth in the normal condition.
ΔSNF1 mutant strain showed moderate defective phenotype under heat shock stress conditions (Fig7C) when compared to the wild type BY4741 strain in 2% glucose-SC medium(Fig7A); however, both strains grew similar at a normal growth temperature of 30°C in 2% glucose-SC medium (7A). Our team also wanted to determine whether the presence of brown algae extracts could make the ΔSNF1 mutant strain at 37°C grow like wildtype BY4741 at 37°C or facilitate growth under normal temperature (no heat shock-induced) of 30°C.
In the presence of 100μg/ul brown algae extract, ΔSNF1 mutant strain still showed moderate defective phenotype at 37°C (Fig7D) when compared to the strain without brown algae extracts at 37°C (Fig7C). BY4741 wild-type strain and ΔSNF1 mutant strain also grew independent to the existence of 100μg/μl brown algae extract as the strain under 30°C in 2% glucose-SC medium with 100μg/μl brown algae (Fig7B) grew similarly to the strain under 30°C in 2% glucose-SC medium without 100μg/μl brown algae (Fig7A).
Overall, the data indicates that the presence of 100μg/μl brown algae extract didn’t alleviate the mild defective phenotype of ΔSNF1 mutant strain at heat shock single stress (7D), nor improve the growth under normal conditions (7B).
Fig 8: Brown algae extract itself, didn’t alleviate either single stress of heat shock, carbon deprivation, or a combination of both on the cell viability plates.
To further determine if brown algae extract could mitigate either carbon deprivation stress or heat shock stress, or both in the environment, an experiments was designed and performed, shown in figure 8.
In the normal condition plate (Fig8A), wildtype 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 condition, wildtype 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% galactose-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.
In Fig8C, the team spread 100ul of 100μg/μl brown algae extracts onto a plate containing 2% galactose-SC, yet wildtype BY4741 and ΔSNF1 remained to exhibit severe growth defective phenotypes similar to those previously mentioned in Fig8B. This observation suggests that brown algae extracts alone don’t mitigate defective phenotype induced by carbon deprivation stresses.
Our team also conducted double stresses on the growth medium plate, with the combination of heat shock stress and carbon deprivation stress induced simultaneously on the yeasts inoculated on the growth medium plate, shown in Fig8E. The growth defective phenotype induced by the double stress was even more remarkable on both wildtype BY4741 and ΔSNF1 strains. The ΔSNF1 strain barely survived on the plate shown in Fig8E, the double stress plate, indicating the double stress combination not only causes ΔSNF1 mutant strain but also wildtype BY4741 to exhibit more significant growth defective phenotypes compared to the yeast strains under single stress conditions shown in Fig8B and Fig8D.
To determine whether brown algae extracts alone could reduce either stresses of the double stress combination or both, 100μg/μl of brown algae extract was used in Fig8C and Fig8F, which showed low mitigation on the growth defective phenotype induced by stresses for both wildtype BY4741 and ΔSNF1 strains when compared to Fig8B and Fig8E.
Overall the data indicates the presence of 100μg/μl brown algae extract alone didn’t alleviate any single stress of heat shock and carbon deprivation or double stress of both combined.
To determine whether our overexpression of SNF1 related genes(N-truncate, C-truncate, 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. There are four composite parts pGal1, 10-SNF1(FL)-SBP (BBa_K4180005), pGal1, 10-SNf1 Δ2-306aa-SBP (BBa_K4180006) , pGal1, 10-SNF1Δ381-633aa -SBP (BBa_K4180007), and pGal1, 10-eGFP-SBP (BBa_K4180008) that are transformed into BY4741 yeast strain. BBa_K4180008 in BY4741 would be used as control as GFP has no function in BY4741 wildtype yeast strain. BBa_K4180006 and BBa_K4180007 are truncation of the SNF1 full length gene, which deletes 2-306 amino acid and 381-633 amino acid for BBa_k4180006 and BBa_K4180007 respectively. The truncated parts are called dominant negative proteins, and deleting this part from the full length gene might interfere with the normal SNF1 protein’s function. The purpose of truncating SNF1 is to determine the domain that causes more severe defective phenotypes.
Fig 9: 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.
On Fig 9, our team showed single stress on Fig9B, carbon deprivation, and Fig9C,heat shock, and double stress on Fig9D. Under heat shock stress (Fig9C), pGal-eGFP in By4741 and pGal-snf1 Δ2-306aa in BY4741 showed slight growth of defective phenotypes compared to normal conditions (Fig9A). pGal-snf1 Δ2-306aa in BY4741showed dramatic growth defective phenotype under heat shock single stress (Fig9C) compared to Fig9A under normal conditions. pGal-snf1Δ381-633aa in BY4741 yeast didn’t show any strong growth defective phenotype in both Fig9C and Fig9A.
To determine whether the combination of brown algae extract and overexpressed downstream genes on our cloned pGal-driven plasmids have any synergistic effect in mitigating defective phenotypes on the cell viability plates under stress conditions, our team spread 100μg/μl Brown algae extracts on yeast growth medium plates on the plates with heat shock single stress. In the presence of 100μg/μl brown algae extract under normal conditions (Fig9B), pGal-snf1 Δ2-306aa in BY4741 moderately reduced the growth defective phenotype compared to Fig9A, yeast strains growing under normal conditions without 100μg/μl brown algae extracts. pGal-snf1 Δ2-306aa in BY4841 also mitigated the growth defective phenotype induced by heat shock single stress when comparing Fig9D, heat shock single stress with 100μg/μl brown algae extracts, and Fig9C, heat shock single stress without 100μg/μl brown algae extracts. However, pGal-eGFP and pGal-SNF1(FL) in BY4741 in heat shock with 100μg/μl brown algae extract (in Fig9D) did not show any alleviation on growth defective phenotype when compared to normal conditions without 100μg/μl brown algae extract (Fig9A).
Overall, the data showed the addition 100μg/μl brown algae extracts with overexpression of pGal-snf1 Δ2-306aa in BY4741 did have a synergistic effect to mitigating its own growth defective phenotype and also the defective phenotype induced by heat shock stress.
Fig10 : Brown algae extracts combined with overexpression of our cloning parts, pGal-SNF1(FL), pGal-snf1 Δ2-306aa , pGal-snf1 Δ381-633aa , and pGal-eGFP 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.
To determine whether brown algae extract could mitigate defective phenotype induced by either stresses or both, the experiment designed and performed is shown in Fig10. Under normal conditions (Fig10A), pGal-eGFP in BY4741 and pGal-SNF1 (FL) in BY4741 respectively, showed normal similar growth phenotypes. However, pGal-snf1 Δ2-306aa in BY4741 strain showed moderate growth defective phenotype when compared to pGal-eGFP in BY4741 and pGal-SNF1(FL) in BY4741 on the same plate (Fig10A). pGal-snf1 Δ381-633aa in BY4741 did not show any strong growth phenotypes on any plates, including the normal control plate shown in Fig10A.
In the presence of carbon deprivation single stress(Fig10B), all strains showed drastic severe growth defective phenotypes when compared to normal conditions(Fig10A). Carbon deprivation single stress (Fig10B) had a stronger impact on all yeast strains when compared to heat shock single stress (Fig10D).
By exposing 100μg/μl brown algae extract on carbon deprivation single stress, showed in Fig10C, all strains remained to show severe growth defective phenotypes similar to those shown in Fig10B, which indicate the presence of 100μg/μl brown algae extract combined with the overexpression of downstream genes on the team’s pGal-driven plasmid doesn’t exhibit any strong synergistic effects to reduce carbon deprivation stress.
Alls strains’ growth defective phenotypes were still drastically exhibited under double stress conditions, shown in Fig10E. The drastic growth defective phenotype shown in Fig10E cannot by mitigated by the addition of 100μg/μl brown algae extracts shown in Fig10F.
Overall, the data indicates that brown algae extract combined with overexpression of downstream genes related to SNF1 of pGal did not have strong synergistic in mitigating defective phenotype induced by carbon deprivation single stress and double stress.
Improvements:
The first improvement for our experiment would be to increase the number of trials we did for the survival plates. Due to the limited time our team had with the lab, we were only able to conduct one trial on the survival plates. Therefore, the data collected may not be an accurate representation of how the BY4741 yeast strain reacts to stresses as well as the addition of nutrients.
Furthermore, our team only looked at day 1 data for our survival plates due to time issues. This may result in missing significant differences in the growth of the yeast that would have occurred down the line. Therefore, an improvement would be to repeat the survival plates and observe the yeast growth data for multiple days.
Another improvement is to demonstrate our survival plates with the addition of green tea extracts. Our team discovered the green tea nutrient extracts are delivered in solid form, which causes difficulties for us to spread them on our plates since the green tea extracts have low solubility to the solvents we have tried, This inhibited us from conducting survival plates with the presence of green tea extracts. In addition to this difficulty our team discovered, there wasn’t enough time for our team to redo the experiment with green tea extract. Hence, we left out the addition of green tea extracts on our survival plates.
Conclusion:
Overall, the data collected indicate that brown algae extract combined with overexpression genes related to SNF1, downstream of a pGal-driven plasmid, doesn’t exhibit strong synergistic effects in mitigating carbon deprivation stress or the combination of both heat shock and carbon deprivation stresses (shown in Fig10); however, brown algae extracts combined with the overexpression of pGal-snf1 Δ2-306aa in BY4741 did mitigate the growth defective phenotype of pGal-snf1 Δ2-306aa in BY4741 induced by heat shock single stress (shown in Fig9).
The conclusion drawn about our collected data further demonstrates that the design of our survival plates and chosen genes had been tested and showed a possible linkage of brown algae working to mitigate environmental stresses. However, further research is required to test the effectiveness of our product with green tea and other supplements from local products in Taiwan proven to have anti-obesity and metabolism-boosting properties to strengthen the validity of our test.
BBa_K4180008 was used as a control for BBa_K4180005, BBa_K4180006, BBa_K4180007 composite parts transformed into BY4741 yeast strain, respectively. The purpose of doing a time course in the presence of galactose was to determine whether our composite part could be induced by the galactose. In the presence of galactose, the control GFP maximum induction showed at least 20-fold induction at 41hr, which clearly showed the BBa_K4180001 basic part could be manipulated easily by the presence of galactose. However, BBa_K4180006 composite site was SNF1 N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation took place on Thr210 to activate the catalytic activity of SNF1, cloned downstream of the Gal1, 10 promoter; BBa_K4180005 composite site was SNF1 full-length coding region cloned downstream of the Gal1, 10 promoter; BBa_K4180007 composite site was SNF1 C-terminus truncated from amino acid381 to 633 deleting the autoinhibitory domain and SIP-interacting domain (SIR) cloned downstream of the Gal1, 10 promoter, three composite sites didn’t show strong manipulation in the presence of galactose. There are several possibilities as to what might have occurred; first, it’s possible that BBa_K4180005, BBa_K4180006, and BBa_K4180007 coding regions were induced before 17 hr of the presence of galactose, which our team didn’t observe. The second possibility was that it’s possible that BBa_K4180005, BBa_K4180006, and BBa_K4180007 coding regions might have interfered with the endogenous SNF1induction in the presence of galactose to cause the SNF1 induction’s suppression.
Results for Yeast Survival Plates
The null mutation of SNF1 in yeast, Δsnf1 was sensitive to environmental stresses such as glucose deprivation, heat shock, and alkaline PH, and ROS stress and metal stress et al., (Hedbacker and Carlson 2008, Casamayor, Serrano et al. 2012) so due to the time sensitivity to not be able to get Δhsp70 as heat shock control on time, our team decided to use Δsnf1 in BY4741 as glucose deprivation and heat shock stresses as a control strain.
Stress induced in the environment by incubating plates at 37°C (heat shock stress) and by replacing glucose in the supplements with galactose (Carbon deprivation), not only induced stress on yeast, but also induced the downstream genes we cloned on the pGal driven plasmid transformed into BY4741. By manipulating heat shock and carbon deprivation respectively and simultaneously on yeast growth medium plates, single stress and double stresses was induced, which mimics the conditions the human body experiences.
The cloned composite sites, BBa_K4180005, BBa_K4180006, and BBa_K4180007, transformed in BY4741 yeast, along with ΔSNF1 in BY4741 were used to demonstrate the cell viability assay (15) on growth medium plates induced with either carbon deprivation, heat shock, or both. The results of these strains was compared with BBa_K4180008 and wild type BY4741.
Fig7: Brown algae extract itself didn’t alleviate heat shock stress nor improve growth in the normal condition.
ΔSNF1 mutant strain showed moderate defective phenotype under heat shock stress conditions (Fig7C) when compared to the wild type BY4741 strain in 2% glucose-SC medium(Fig7A); however, both strains grew similar at a normal growth temperature of 30°C in 2% glucose-SC medium (7A). Our team also wanted to determine whether the presence of brown algae extracts could make the ΔSNF1 mutant strain at 37°C grow like wildtype BY4741 at 37°C or facilitate growth under normal temperature (no heat shock-induced) of 30°C.
In the presence of 100μg/ul brown algae extract, ΔSNF1 mutant strain still showed moderate defective phenotype at 37°C (Fig7D) when compared to the strain without brown algae extracts at 37°C (Fig7C). BY4741 wild-type strain and ΔSNF1 mutant strain also grew independent to the existence of 100μg/μl brown algae extract as the strain under 30°C in 2% glucose-SC medium with 100μg/μl brown algae (Fig7B) grew similarly to the strain under 30°C in 2% glucose-SC medium without 100μg/μl brown algae (Fig7A).
Overall, the data indicates that the presence of 100μg/μl brown algae extract didn’t alleviate the mild defective phenotype of ΔSNF1 mutant strain at heat shock single stress (7D), nor improve the growth under normal conditions (7B).
Fig 8: Brown algae extract itself, didn’t alleviate either single stress of heat shock, carbon deprivation, or a combination of both on the cell viability plates.
To further determine if brown algae extract could mitigate either carbon deprivation stress or heat shock stress, or both in the environment, an experiments was designed and performed, shown in figure 8.
In the normal condition plate (Fig8A), wildtype 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 condition, wildtype 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% galactose-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.
In Fig8C, the team spread 100ul of 100μg/μl brown algae extracts onto a plate containing 2% galactose-SC, yet wildtype BY4741 and ΔSNF1 remained to exhibit severe growth defective phenotypes similar to those previously mentioned in Fig8B. This observation suggests that brown algae extracts alone don’t mitigate defective phenotype induced by carbon deprivation stresses.
Our team also conducted double stresses on the growth medium plate, with the combination of heat shock stress and carbon deprivation stress induced simultaneously on the yeasts inoculated on the growth medium plate, shown in Fig8E. The growth defective phenotype induced by the double stress was even more remarkable on both wildtype BY4741 and ΔSNF1 strains. The ΔSNF1 strain barely survived on the plate shown in Fig8E, the double stress plate, indicating the double stress combination not only causes ΔSNF1 mutant strain but also wildtype BY4741 to exhibit more significant growth defective phenotypes compared to the yeast strains under single stress conditions shown in Fig8B and Fig8D.
To determine whether brown algae extracts alone could reduce either stresses of the double stress combination or both, 100μg/μl of brown algae extract was used in Fig8C and Fig8F, which showed low mitigation on the growth defective phenotype induced by stresses for both wildtype BY4741 and ΔSNF1 strains when compared to Fig8B and Fig8E.
Overall the data indicates the presence of 100μg/μl brown algae extract alone didn’t alleviate any single stress of heat shock and carbon deprivation or double stress of both combined.
To determine whether our overexpression of SNF1 related genes(N-truncate, C-truncate, 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. There are four composite parts pGal1, 10-SNF1(FL)-SBP (BBa_K4180005), pGal1, 10-SNf1 Δ2-306aa-SBP (BBa_K4180006) , pGal1, 10-SNF1Δ381-633aa -SBP (BBa_K4180007), and pGal1, 10-eGFP-SBP (BBa_K4180008) that are transformed into BY4741 yeast strain. BBa_K4180008 in BY4741 would be used as control as GFP has no function in BY4741 wildtype yeast strain. BBa_K4180006 and BBa_K4180007 are truncation of the SNF1 full length gene, which deletes 2-306 amino acid and 381-633 amino acid for BBa_k4180006 and BBa_K4180007 respectively. The truncated parts are called dominant negative proteins, and deleting this part from the full length gene might interfere with the normal SNF1 protein’s function. The purpose of truncating SNF1 is to determine the domain that causes more severe defective phenotypes.
Fig 9: 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.
On Fig 9, our team showed single stress on Fig9B, carbon deprivation, and Fig9C,heat shock, and double stress on Fig9D. Under heat shock stress (Fig9C), pGal-eGFP in By4741 and pGal-snf1 Δ2-306aa in BY4741 showed slight growth of defective phenotypes compared to normal conditions (Fig9A). pGal-snf1 Δ2-306aa in BY4741showed dramatic growth defective phenotype under heat shock single stress (Fig9C) compared to Fig9A under normal conditions. pGal-snf1Δ381-633aa in BY4741 yeast didn’t show any strong growth defective phenotype in both Fig9C and Fig9A.
To determine whether the combination of brown algae extract and overexpressed downstream genes on our cloned pGal-driven plasmids have any synergistic effect in mitigating defective phenotypes on the cell viability plates under stress conditions, our team spread 100μg/μl Brown algae extracts on yeast growth medium plates on the plates with heat shock single stress. In the presence of 100μg/μl brown algae extract under normal conditions (Fig9B), pGal-snf1 Δ2-306aa in BY4741 moderately reduced the growth defective phenotype compared to Fig9A, yeast strains growing under normal conditions without 100μg/μl brown algae extracts. pGal-snf1 Δ2-306aa in BY4841 also mitigated the growth defective phenotype induced by heat shock single stress when comparing Fig9D, heat shock single stress with 100μg/μl brown algae extracts, and Fig9C, heat shock single stress without 100μg/μl brown algae extracts. However, pGal-eGFP and pGal-SNF1(FL) in BY4741 in heat shock with 100μg/μl brown algae extract (in Fig9D) did not show any alleviation on growth defective phenotype when compared to normal conditions without 100μg/μl brown algae extract (Fig9A).
Overall, the data showed the addition 100μg/μl brown algae extracts with overexpression of pGal-snf1 Δ2-306aa in BY4741 did have a synergistic effect to mitigating its own growth defective phenotype and also the defective phenotype induced by heat shock stress.
Fig10 : Brown algae extracts combined with overexpression of our cloning parts, pGal-SNF1(FL), pGal-snf1 Δ2-306aa , pGal-snf1 Δ381-633aa , and pGal-eGFP 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.