Team:Tunghai TAPG/Collaborations

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Collaboration with 2019 Mingdao iGEM 1

There's an iGEM team cooperation from Ming Duo Senior High School. We realized they want to invent a machine which can absorb carbon dioxide. We prepared a course for them to understand what kind of methods to absorb the carbon dioxide in chemical industry. We are really glad that we can use our knowledge to provide some helps.

How do we capture carbon dioxide ?

There are three main approaches to capture carbon dioxide . First, capture it after burning fuel, secondly, capure it before the fuel is burned, third, burn fuel in such a way that the carbon is easy to capture.

  1. Capture carbon dioxide after burning fuel: In a post-combustion method, CO2 is removed after combustion of fossil fuel. The exhaust (or flue) gases from power station are mainly nitrogen. Only about 15% of the exhaust gases is CO2. The absorption towers would replace smokestacks where the CO2 would be taken out of the emissions by chemicals called amine.The second column, known as a stripper.It strips CO2 to leave the system so that amine could be used again.
  2. Capture carbon dioxide before burning fuel: In one of the pre-combustion methods, fossil fuel is oxidized before it is burned. This way produces “syngas” which is made of carbon oxide and hydrogen. The resulting carbon emissions can be pulled off in a relatively pure stream while the hydrogen is burned as fuel.
  3. Make carbon dioxide to be captured easily: In Oxy-fuel combustion, the fuel is burned in pure oxygen instead of air. The water vapor condenses through cooling. This process produced an almost pure stream of CO2 that can be transported to the sequestration site and to be stored,that's how the CO2 captures work.

After the collaboration with us ,we showed them around our campus and also introduced to them our church which is really famous.The Luce Chapel at Tunghai University was the first example of modernist architecture in Taiwan. Created by Chinese-American architect I. M. Pei and Taiwanese architect Chen Chikwan, it is considered to be one of the most distinctive buildings of 20th century architecture around the globe.

Collaboration with 2019 Mingdao iGEM 2

Tunghai_TAPG

We lent Team Mingdao our chemical laboratory and also provided them with some suggestions about their experiment, which was about using benzene to measure their enzyme, CYP2E1’s efficiency. We discussed with their team about the method of analyzing the result of their measurement.

Collaboration with 2019 Mingdao iGEM 3

Characterization of Fluorescence intensity

We provided them the transformed E. coli BL21(DE3) with BioBricks and growth media (LB+Kan medium). Team Mingdao assisted us in measuring the OD values of the E. coli with IPTG addition for confirming the IPTG induction of the gene expression.

Protocol

  1. Culture E. coli BL21(DE3) carrying vector only or T7-RBS-EGFP in LB + Kan (50 μg/mL) O/N at 37°C
  2. Measure OD600
  3. Dilute to OD600 around 0.1
  4. Shake at 200 rpm, 37°C until OD600 around 0.8
  5. Add 1mM IPTG for protein inductio
  6. Measure OD600 and Ex/Em=488/518 nm every 30min for 24hr

Result

The TAPG tunghai team 2019 has constructed a fusion protein by using our peptide JJ01’s back end combined with eGFP, which was induced by IPTG for expression, to ensure the accuracy of our protein expression. The fluorescent protein was employed as the identification, indicating the yield of our protein. Among of these, we have investigated four different cells in total (normal BL21, BL21 with fluorescent protein, BL21 adding IPTG, BL21 with fluorescent protein adding IPTG), and designed three strategies to ensure the accuracy.

In Figure 1. the x-axis refers to time, the y-axis refers to OD600 value. We measured the OD600 of four cells during the experiments, continually to 24 hours, and plotted the growth curve. Apparently, there is no obvious gap among these four cells.

In Figure 2. the x-axis refers to time, the y-axis refers to Fluorescence intensity. We then quantified four cells’ Fluorescence intensity, individually (Ex/Em=488/518nm), we could observe that, the curves of two cells without eGFP were not overlapping, and at the very bottom of the chart. As for the cell with eGFP but without IPTG induction, the curve remained horizontal and sended out few fluorescent. When IPTG are adding in the cell with fluorescent, we could notice that the curve has an obvious change. After adding 1 mM of IPTG, while OD600 reached to 0.8, the curve displayed a dramatic growth.

In Figure 3. the x-axis refers to time, the y-axis refers to Fluorescence intensity. We divided Figure 2. by Figure 1. and got the Fluorescence intensity of four different kind of cells per unit; then we were able to compare the productions correctly by knowing Fluorescence intensity from each cells. Because the quantity of Fluorescence protein was proportional to the target gene that was expressed. In order to know the Fluorescence intensity in each bacteria, we have to divide the numbers of bacteria by total Fluorescence intensity, which would provide us the quantity of the Fluorescence intensity per bacteria.


Figure.1 OD600

Figure.2 Ex/Em=488/518 nm

Figure.3 Ex/Em=488/518 nm normalized with OD600

Conclusion

Team Mingdao helped us to find the actual ability of IPTG inhibiting the production of EGFP. We’re glad to cooperate with Team Mingdao, which is very experienced in iGEM competition, giving us lots of suggestions about our project. Hope we can still maintain the friendly cooperation relationship every year from now on Mingdao, thanks a lot!

Collaboration from the 2019 CSMU iGEM

They hleped us to do the protein overexpression .There's the experiment below. We faced a challenge which is about to express the protein, so we stop our research and discuss it for a couple weeks. Fortunately, we met the CMSU igem which wanted to help us for solving this problem, so we hung out for a couple times to talk about these problems, we can’t complete our project without theirs help.

Day 0

(Turn on mountaineering gas, obtain 2 sterilized 50cc centrifuge tube)

  1. Sterilize the rim of LB bottle, add 15cc sterilized LB to each 50cc centrifuge tube.
  2. Sterilize the rim of pick bottle, and carefully take out one pick (gently shake the bottle first until you are able to take out one without touching the others.)
  3. Pick a colony and stir the pick in the LB.
  4. Add 15ul Kanamycin (50mg/mL) into LB, and vortex to mix evenly.
  5. Turn off the mountaineering gas.
  6. Tight up the lid and label each centrifuge tube.
  7. Incubate in incubator at 37℃, 150 rpm(make sure the centrifuge tube stays tilted at about 45 degrees).
  8. Incubate overnight (12-18hrs).

Day 1

  1. Obtain sterilized 200cc LB in a 500 mL Erlenmeyer.
  2. Add 0.2 mL Kanamycin (50mg/mL) into LB, and vortex to mix evenly.
    3. (Note: Add 1/1000 volume of antibiotic into LB.)
  3. Take out the two tubes of liquid cultures from the incubator.
  4. Take 10 mL from each two cultures and centrifuge for 20mins at 6000rpm.
  5. Remove the supernatant.
  6. Add a little LB from the 200 mL sterilized LB to the two centrifuged tubes, vortex to re-suspend the pellet, and pour them into the 200 mL sterilized LB.
  7. Take 1 mL to test the OD ratio(595nm, ABS).
  8. Incubate in the incubator at 37℃, 150rpm for 2 hours and test the OD ratio. If the ratio is between 0.4 to 0.7, continue the next step. If the ratio is lower than 0.4 keep incubating; if the ratio exceeds 0.7, repeat from step 4 again.

Cell Disruption

  1. Add 100mM IPTG and wait for 5mins under room temperature, then spin down.
  2. Incubate in the incubator for 2.5 hrs.
  3. Allocate the 200 mL LB evenly into 50 mL centrifuge tubes, usually 40 mL*5 tubes.
  4. Add ddH2O until each tube weighs the same.
  5. Centrifuge at 6000rpm for 20mins.
  6. Discard the supernatant, and re-suspend the pellet by adding 4 mL 20mM Tris(pH7.6), vortex.
  7. Mix the re-suspends into a same centrifuge tube. Centrifuge at 6000rpm for 20mins.
  8. Discard the supernatant, re-suspend the pellet by adding 20 mL 20mM Tris (pH7.6), vortex.
  9. Add Protein inhibitor PMSF 0.2 mL.
  10. Cell disruption by Ultrasonic Homogenizer.
  11. Draw 100 μL as sample for total(T), Draw another 100 μL as sample for pellet(P), and the others are collected 1 mL in per eppendorfs.
  12. Centirfuge pellet and the others at 8,700G for 20mins under 4℃
  13. Collect the supernatant of P as S(soup), and re-suspend P with 1 mL 20mM Tris (pH7.6). Collect the supernatant of others in a clean 15 mL centrifuge tube.
  14. Draw 100 μL as sample T1(total 1), Draw another 100 μL as sample P1 (pellet 1) and the others are collected 1 mL in per eppendorf
  15. Centrifuge sample P1(pellet 1) and the others at 16,000G for 20mins under 4℃
  16. Collect the supernatant of P1 as S1, and re-suspend P1 with 1 mL 20mM Tris (pH7.6). Collect the supernatant of others in a clean 15 mL centrifuge tube, this is the target product of our protein expression.
  17. Add 100 μL 2xSB to T, P, S, T1, P1, S1, and heat the sample at 37 ℃ for 10 min.
  18. SDS page or western blot.

Ultrasonic Homogenizer Settings

  1. Active interval 5 secs
  2. passive interval 15secs,150 cycles, active total time 12min 30secs, total operating time 50mins.