Team:Mingdao/Collaborations
Collaborations
Team: NCTU_Formosa
Team: Tunghai_TAPG
Team: CSMU_Taiwan
Collaborations
We collaborated with three collegiate iGEM teams this year, NCTU_Formosa, Tunghai_TAPG, and CSMU_Taiwan, respectively. National Chiao Tung University (NCTU_Formosa) gave us technical support to accomplish the complicated experiments. Tunghai University (Tunghai_TAPG) is the first team we collaborated with this year, and they provided us with some good ideas about our product. We would like to express our special thanks to Chung Shan Medical University (CSMU_Taiwan ) as we only started cooperating with them since last year. It only takes us 10 minutes driving to the CSMU campus, so we usually have weekly meetings with team CSMU_Taiwan. We really have a great season with these teams!
Team: NCTU_Formosa
Go to NCTU_Formosa's wiki to see what they say about our interaction
Helped by NCTU_Formosa
They gave advice on our collaboration and presentation. In addition, they helped us verify the protein expression induced by our experimental procedure. NCTU conducted SDS-PAGE with the Bacillus total lysates prepared by us. The gel stained with coomassie blue was presented below. Lanes 2 & 3 showed CYP2E1 with different cofactors (5-aminolevulinic acid and 5-aminolevulinic acid plus FeSO4, respectively) at ~57 kDa. Due to many other proteins stained similar in size (see Lane 1 for the WT control), although with a thinner band on the gel, the CYP2E1 expression and the protein induction conditions need to be further examined.
Mingdao did a favor for NCTU_Formosa
They transformed E.coli BL21 (DE3) with ydfD/pSB1A3 for the expression of the toxin ydfD in the control of IPTG. In order to test the growth affected by the toxin, we measure the growth curve of the transformed E.coli in the presence or absence of IPTG.
Experiment procedure
↓ culture E. coli BL21(DE3) carrying ydfD/pSB1 in LB + 50 μg/ml of AMP O/N at 37°C
↓ measure OD600
↓ dilute 100x into a total volume of 6 ml of media
↓ shake at 200 rpm at 37°C until OD600 around 0.3
↓ take 200μl of bacterial liquid to 96 well plate
↓ add 1mM IPTG to the rest of bacterial liquids for protein induction
↓ take 200μl of bacterial liquid with 1mM IPTG to 96 well plate
↓ measure OD600 every 10min for 7hr
We picked up 2 clones and did 6 repeats for each group. The data are presented as average numbers from 6 repeats of independent clone.
Result
Clones 1 & 2 grew smoothly as usual without IPTG induction and are set as controls. Clones 1 & 2 with IPTG expressed the toxin of ydfD and grew a little slower than the controls. However, the growth rate speeded up instantly around the OD of 0.7 at the time of 250 min. The result suggested the toxin affects the growth of E. coli and may cause adaptive mutation in E. coli in the pressure of the toxin.
Conclusion
We thank the team NCTU-Formosa for this collaboration. We learned a lot about experiment design, the skills of troubleshooting, and the way to analyze and present the data in a scientific way.
Team: Tunghai_TAPG
Go to Tunghai_TAPG's wiki to see what they say about our interaction
Helped by Tunghai_TAPG
They provided space for benzene operation in Prof. Feng-Di Lung‘s lab in Department of Chemistry, Tunghai University to check the function of protein CYP2E1 and gave suggestions about the VOCs measurements.
We prepared the lysates containing CYP2E1 and designed a measurement method based on 4-aminoantipyrine colorimetric reaction. They helped to conduct benzene analysis in a chemical hood with safety manipulation. Benzene is catalyzed by CYP2E1 and converted to phenol. We would be able to check the concentration of phenol by this method. The result will show the efficiency of CYP2E1.
Experiment procedure
↓ prepare CYP2E1 and WT lysates
↓ prepare 1.79 g/L of benzene (i.e., benzene solubility in water)
↓ add 90μl of benzene solution with 10X serial dilution to each well
↓ incubate with 40μl of CYP2E1 or WT Bacillus lysates at room temperature for 30 min
↓ transfer 90μl of the mixture to a new well
↓ add 90μl of solution I (1% of 4-aminoantipyrine in KOH solution, pH=9~10
↓ then add 45μl of solution II (4% of K3[Fe(CN)6])
↓ measure at OD580
Result
The 4-aminoantipyrine in alkali condition will turn yellow to dark red in the presence of phenol with the oxidative K3[Fe(CN)6], which color can be measure at OD580.
The data showed that the values measured at OD580 are higher in the group of benzene plus CYP2E1 compared to controls of benzene only, suggesting that phenol is converted from benzene by CYP2E1.
Mingdao did a favor for Tunghai_TAPG
We provided the spectrophotometer to detect fluorescence intensity and bacterial growth, as well as giving advice on cloning strategies and protocols to use and wiki design tricks. They prepared the transformed E. coli BL21(DE3) with BioBricks and growth media (LB+Kan medium) and we measured the OD values of the E. coli cultured overnight and added IPTG for the induction of the gene expression.
Experiment procedure
↓ culture E. coli BL21(DE3) carrying vector only or T7-RBS-EGFP in LB + Kan (50 μg/ml) O/N at 37°C
↓ measure OD600
↓ dilute to OD600 around 0.1
↓ shake at 200 rpm, 37°C until OD600 around 0.8
↓ add 1mM IPTG for protein induction
↓ measure OD600 and Ex/Em=488/518 nm every 30min for 24hr
Result
GFP expression is driven by T7 promoter which is under control by IPTG in E. coli BL21(DE3) strain. The fluorescence intensity was detected at basal level in E. coli carrying T7-RBS-EGFP compared to the control groups. The intensity of GFP was induced by IPTG in a time-dependent manner, confirming that IPTG indeed induces the yield of GFP.
Conclusion
It’s our pleasure to collaborate with iGEM team Tunghai_TAPG. We have been cooperating with them since April, and they gave us a lot of advice about our project. Our appreciation to team Tunghai_TAPG is beyond description. Thank you Tunghai_TAPG, hope we can still work together next year!
Team: CSMU_Taiwan
Go to CSMU_Taiwan's wiki to see what they say about our interaction
Helped byTeam: CSMU_Taiwan
We discussed the project several times in the meetings with CSMU_Taiwan. They gave us a lot of valuable advice and always inspired us to view the experimental results in a different way. This year we appreciate being mentored by a collegiate team, CSMU_Taiwan.
In addition, as our project aims for the effect of CA and CYP2E1 addition, they gave us a hand on the purification of carbonic anhydrase. We provided Bacillus subtilis 168 carrying human carbonic anhydrase II with a C-terminal 6xHis tag, and CSMU_Taiwan purified CA for us, which led to better efficiency of CO2 dissolution.
Mingdao did a favor for CSMU_Taiwan
We gave suggestions on their project and experiments and performed gene cloning for them. We are good at skills of TA cloning and gene cloning, and successfully inserted the small DNA fragment (i.e., aptamers) into plasmids, which enabled their project to keep going on.
Conclusion
CSMU_Taiwan was one of our best partners. We constantly visited each other and discussed the experiment results all the time. We even did some experiments together in the lab until midnight. We also encouraged each other to survive in this tough iGEM season.
Quote
“”
Follow us on
Contact us
Phone: +886-4-23341797
EMAIL: biolab@ms.mingdao.edu.tw
No.497, Sec. 1, Zhongshan Rd., Wuri District, Taichung City 414, Taiwan (R.O.C.)