Team:Nanjing-China/Notebook

Home

Journal

In March, we founded our team and recruited our team members, after with we divided our team members into several groups and each group was responsible for one part of the competition. We were also busy brainstorming about our project and signing up for iGEM competition. Besides, we started to help students from Southeast University to help them build a new team.

 

In April, we confirmed our project. A majority of the team members wanted to be focused on hydrogen production. Then we searched online and read many articles related to our project. We also consulted many experts in hydrogen production and discussed our projects with instructors. We also made a plan about the procedures of all the experiments.

 

In May, we were busy with kit construction and part construction. We were also contacting different authorities and scheduled for community activities. We even designed questionnaires to collect public opinion about hydrogen production and our project.

 

In June, we went on with part construction and lab work. We also opened our lab to primary school students and attended conferences in ZJU and met with many other iGEMers in China. Team members from TMMU-China visited us and we gave them many important suggestions. We think it is really important to help new iGEMers!

 

In July, we successfully constructed all our parts and expressed hydrogenase. We began to test the hydrogen production efficiency and explore conditions for CdS precipitation and silicon encapsulation. Meanwhile, we did some human practices such as community propagation, interviews and collaborations with other iGEMers!

 

In August, we still spared no efforts to continue lab work and also attended the CCiC conference in Guangzhou and presented our project to Chinese iGEM teams.

 

In September, we were engaged in some details of lab work and were invited by TMMU-China for a conference. We are also busy with wiki construction, logo design and visa application.

 

In October, everyone is busy with the project, from lab work, data analysis, writing staff, modeling to human practices. We also rehearse our presentation several times before going to Boston.

Protocols

 

DNA purification/Axygen gel extraction

 

1. Excise the agarose gel slice containing the DNA fragment of interest with a clean, sharp scalpel under ultraviolet illumination.

 

2. Absorb the liquids left on the surface of the gel slices using paper towels. Weigh gel slice (tare with empty tube).

 

3. Add 3 volumes of DE-A buffer per mg of gel (so a 100mg gel gets 300ul of buffer).

 

4. Resuspend the gel in Buffer DE-A by vortexing. Heat at 75℃ until the gel is completely dissolved (keep heating for 6-8 minutes). If low-melt agarose gel is used, you may heat it at 40℃. Intermittently vortexing every 2-3 minutes will do a lot of help to accelerate the solubilization.

 

Note: Buffer DE-A is red liquid, so you can observe the color to make sure the gel is fully dissolved.

 

5. Add 0.5× Buffer DE-A volume of Buffer DE-B and mix. If the DNA fragment is less than 400bp, supplement further with a 1×sample volume of isopropanol.

 

Note: After the addition of DE-B, the solution should be in the uniform color of yellow.

 

6. Place a Miniprep column into a 2ml microfuge tube (provided). Transfer the solubilized agarose from the step above into the column. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

7. Return the Miniprep column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.

 

8. Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 30 seconds. Discard the filtrate from the 2ml microfuge tube.

 

9 Place the Miniprep column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

10. Place the Miniprep column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.

 

11. Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 50ul of ddH2O to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.

 

Note: Pre-warm the ddH2O at 65℃ will generally improve elution efficiency.

 

 

Preparation of chemically competent E.coli cells

 

1. Inoculate 2ml LB broth with an aliquot (about 50ul)of the desired E.coli from the -80℃ freezer stock of cells.

 

2. Incubate for 2h at 37℃.

 

3. Add the 2ml seed culture to 250ml LB broth and grow at 37℃, shaking (about 200rpm) until OD600 of 0.3-0.4 (about 5 hours).

 

4. Pre-cool the 50ml polypropylene tube, 80 EP tubes, CaCl2-glycerine (0.1mol/L CaCl2) and CaCl2- MgCl2 (80mmol/L MgCl2, 20mmol/L CaCl2). Set the centrifuge and prepare the ice tray.

 

5. Transfer the bacteria into the 50ml polypropylene tube. Place it on ice for 10 minutes.

 

6. Centrifuge at 4℃, 4100rpm for 10 minutes.

 

7. Discard supernatant, then place the tube upside down to make sure trace liquid medium runs out.

 

8. Add 30ml of pre-cooled CaCl2- MgCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.

 

9. Centrifuge at 4℃, 4100rpm for 10 minutes.

 

10. Discard supernatant then place the tube upside down to make sure trace liquid medium runs out.

 

11. Add 2ml of pre-cooled CaCl2 per 50ml of initial liquid medium to resuspend bacteria cell pellet.

 

12. Transfer to EP tubes (50ul every tube) and store at -80℃.

 

 

General Heat-Shock Transformation

 

1. Add 10ul DNA to 50ul cells on ice (set positive control by using Pcotc, cotc, PtasA, GolB,PbrR DNA fragment and ddH2O, set negative control by using chemically competent E.coli cells without plasmids).

 

2. Incubate on ice for 30 minutes.

 

3. Heat shock at 42℃ for exactly 90 seconds.

 

4. Place samples back on ice for 1-2 minutes.

 

5. Operating in the clean bench, add 900ul of LB broth per tube.

 

6. Incubate at 37℃ for 60 minutes, shaking.

 

7. Activate it on the plate for 60 minutes. The total number of plates is 7.

 

8. Centrifuge at 3000rpm for 1 minute.

 

9. Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.

 

10. Use the inoculating loop to load bacteria liquid then streak on the LB plate.

 

11. Place plates upside down and incubate at 37℃ overnight.

 

 

PCR method/Taq PCR

 

1. Thaw Taq, dNTP, primers, template DNA (pcotc, cotc, PtasA, tasA, GolB, PbrR) on ice.

 

2. To a new PCR tube, add:

 

template DNA 1ul
dNTP 1ul
10×buffer 5ul
Mg2+ 3ul
F primer 1ul
P primer 1ul
rTaq E 1ul
ddH2O 37ul
total 50ul

 

3. Mix solution well.

 

4. Place tube in PCR thermocycler. Set thermocycler program:

 

Inititial denaturation: 3min at 95℃;

 

Loop (29 cycles),
Denaturation: 30s at 95℃,

 

Annealing: 30s at 60℃,

 

Elongation: 1min at 72℃;

 

Final elongation: 10min at 72℃;

 

Store: 12℃.(not for too long).

 

5. We use 5ul of the PCR product for electrophoresis and 45ul for purification (details see DNA purification/AxyPrep PCR DNA purification PCR).

 

 

DNA purification/AxyPrep PCR DNA purification PCR

 

1. Add 3 volumes of Buffer PCR-A to the solution (if Buffer PCR-A is less than 100ul, then add to 100 ul). Mix gently and then transfer to a Miniprep column, which is placed in a 2ml microfuge tube (provided).

 

2. Centrifuge at 12,000rpm for 1 minute and discard the filtrate from the 2ml microfuge tube.

 

3. Return the Miniprep column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

4. Return the Miniprep column to the 2ml microfuge tube and add 400ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

Note: this step can be omitted.

 

5. Transfer the Miniprep column into a clean 1.5ml microfuge tube (provided). Add 25-30ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute.

 

Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.

 

 

Plasmid extraction

 

1. Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely.

 

2. Add 250ul of Buffer S1 to the pellet to resuspend bacteria cells.

 

3. Add 250ul of Buffer S2, mix gently by inverting the tube 4-6 times until the solution becomes clear. The time should be no longer than 5 minutes.

 

4. Add 350ul of Buffer S3, mix gently by inverting the tube 6-8 times.

 

5. Centrifuge at 12,000rpm for 10 minutes.

 

6. Place spin column into a 2ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

7. Return the column to the 2ml microfuge tube and add 500ul of Buffer W1. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

8. Return the column to the 2ml microfuge tube and add 700ul of Buffer W2. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

9. Place the column back into the 2ml microfuge tube. Add a second 700ul of Buffer W2 and centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.

 

10. Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 1 minute.

 

11. Transfer the column into a clean 1.5ml microfuge tube (provided). Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Note: Pre-warm the Eluent or deionized water at 65℃ will generally improve elution efficiency.

 

 

Agarose Gel Electrophoresis

 

1. Weigh agarose powder and TAE buffer according to a proper portion, and add them to a 100ml conical flask (we usually make 1.5% Agarose Gel).

 

2. Melt the mixture in a microwave until the solution becomes clear (don’t leave the microwave).

 

3. Let the solution cool down to about 40-50℃ and add DNA gel stain (usually we use EB), pour the solution into the gel casting tray with appropriate comb.

 

4. Let the gel cool until it becomes solid.

 

5. Pull out the comb carefully.

 

6. Place the gel in the electrophoresis chamber.

 

7. Add enough TAE Buffer so that there is about 2-3mm of buffer over the gel.

 

8. Pipette DNA samples mixed with appropriate amount of DNA loading buffer (the dye/GeneFinder is in the loading buffer) into wells on the gel.

 

9. Run the gel at 135V for about twenty minutes.

 

 

Colony PCR

 

1. We carry out colony PCR in order to amplify a few copies of DNA across several orders of magnitude and check the length of DNA sequences between two designed primers.

 

2. To 20 new PCR tubes (add 2ul bacteria cells and operate thermal cracking at 95℃ for 15 minutes), add:

 

template DNA 2ul
dNTP 1ul
10×buffer 5ul
Mg2+ 3ul
F primer 1ul
P primer 1ul
rTaq E 1ul
ddH2O 36ul
total 50ul

 

3. To 2 new PCR tubes (tasA, pbrR), add:

 

template DNA 1ul
dNTP 1ul
10×buffer 5ul
Mg2+ 3ul
F primer 1ul
P primer 1ul
rTaq E 1ul
ddH2O 37ul
total 50ul

 

4. Mix solution well.

 

5. Place tube in PCR thermocycler. Set thermocycler program:

 

Loop (29 cycles),
Denaturation: 30s at 95℃,

 

Annealing: 30s at 60℃,

 

Elongation: 1min at 72℃;

 

Final elongation: 10min at 72℃;

 

Store: 12℃.(not for too long).

 

6. use the PCR product for electrophoresis.

 

 

Protocol for restriction enzyme digestion

 

To a 1.5ml microfuge tube (Ptt, Pcc), add:

 

Insert 17ul
bamHⅠ 1ul
KpnⅠ 1ul
10×K 1ul
total 20ul

 

 

Protocol for DNA ligation

 

1. To a 1.5ml microfuge tube (Ptt), add:

 

Vector 1ul
Insert 7ul
T4 Buffer 1ul
T4 Ligase 1ul
total 10ul

 

2. To a 1.5ml microfuge tube (Ptt control), add:

 

Vector 1ul
ddH2O 7ul
T4 Buffer 1ul
T4 Ligase 1ul
total 10ul

 

3. To a 1.5ml microfuge tube (Pcc), add:

 

Vector 1ul
Insert 7ul
T4 Buffer 1ul
T4 Ligase 1ul
total 10ul

 

4. To a 1.5ml microfuge tube (Pcc control), add:

 

Vector 1ul
ddH2O 7ul
T4 Buffer 1ul
T4 Ligase 1ul
total 10ul

 

5. Incubate at 16℃.

 

 

PbrR protein’s inducing / CdS nanoparticle’s in-situ synthesis

 

1. For the engineered-competent E.coli cells stored at -80℃, culture them on LB-agar medium using streak plate method.

 

2. After culturing on LB-agar at 37℃ for 12 hours, pick a single colony, shake in 3mL LB liquid buffer, 37℃, 160rpm, for 8 hours over night.

 

3. Extract 1mL from 300mL sanitized LB liquid buffer, measure its OD600 (Eppendorf TM Biophotometer plus) as blank. Add the 3mL product of step ii into ~300mL LB liquid buffer.

 

4. Shake for about 2 hours, 37℃, 160rpm, until OD600 reaches ~0.6 (OD600 should be measured after 1.5 hours and could be predicted using the formula OD600t+20min=2*OD600t).

 

5. Add 300uL 20% arabinose into the ~300uL product of step iv. Shake at the same condition as before for 1h, inducing the expression of PbrR protein.

 

6. Add 30uL 1M Cd(NO3)2 into the ~300mL product of step v. Keep the initial Cd2+ concentration at 0.1mM. Shake at the same condition for 4h, letting PbrR express adequately and absorb Cd2+ fully.

 

7. Add 30uL 1M NaS2 into the ~300mL product of step vi. Keeping the initial concentration of S2- at 0.1mM.

 

8. Shake at the same condition for 1h, letting S2- captured by Cd2+/PbrR complex, forming the core of the lattice and finally grow to be CdS nanocrystal.

 

 

PDADMAC and PSS’s layer-by-layer assembly and catalyze the dehydration of silica acid to finish encapsulation.

 

1. Harvest the ~300mL product of last step 8 by centrifuge at 3000rpm for 10min (using EppendorfTM Centrifuge 5430R and 6* CorningTM 50mL centrifuge tube).

 

2. Discard the supernatant, re-suspend the sediment with ~5mL 10% NaCl aqueous. Collect the E.coli suspension into a single CorningTM 50mL centrifuge tube.

 

3. Centrifuge again (3000rpm, 10min) and wash again with ~30mL 10% NaCl aqueous. Centrifuge for the third time to harvest all the clean E.coli with CdS nanocrystal.

 

4. Add 20mL 5g/L PDADMAC (poly dimethyl diallyl ammonium chloride, diluted from 20%wt in H2O, average molecular weight 100000~200000, Sigma-AldrichTM) into the sediment gained from step iii. Gently re-suspend to assemble the first layer, react 5min.

 

5. Centrifuge (3000rpm, 5min) to separate E.coli from PDADMAC, discard the supernatant. Wash with 20mL 10% NaCl aqueous, centrifuge (3000rpm, 5min) again and discard the supernatant.

 

6. Add 20mL 5g/L PSS (poly sodium p-styrenesulfonate, diluted from 30%wt in H2O, Sigma-AldrichTM) into the sediment gained from step v Gently re-suspend to assemble the second layer, react 5min.

 

7. Centrifuge (3000rpm, 5min) to separate E.coli from PSS, discard the supernatant. Wash with 20mL 10% NaCl aqueous, centrifuge (3000rpm, 5min) again and discard the supernatant.

 

8. Repeat step iv~vii for 10 times to form a 20-layer structure (10* repetition of PDADMAC+PSS).

 

9. Repeat step iv~v to assemble the last layer, for it ought to be the positive-charged PDADMAC.

 

10. Add 50mL PBS with silica acid (0.1M K2HPO4 : 0.1M KH2PO4 : Na2SiO3 = 800mL : 200mL : 0.01mol) into the sediment gained from step ix, i.e. washed by NaCl aqueous. Gently re-suspend to encapsulate the silica oxide shell. Shake (160rpm, r.t.) for 1h.

 

11. Centrifuge (4000rpm, 10min) to harvest E.coli@SiO2 with CdS nanocrystal.

 

 

Hydrogenase inducing

 

1. Prepare IPTG (1M)、IPTG (1 M)、(NH4)Fe(SO4)2 (1 M)、NiCl2 (1 M) sterile solution.

 

2. If it is encapsulated E.coli with pet28a-hyaABCDEF, get some LB medium to resuspend. If it is normal E.coli with OD600=0.6 or E.coli with pet28a-hyaABCDEF and precipitated CdS, centrifuge at 4000rpm for 10min and add LB to resuspend.

 

3. Add at V(1:1000) of IPTG (1 M)、(NH4)Fe(SO4)2 (1 M)、NiCl2 (1 M), make the final concentration of them to be 1mM.

 

4. Incubate at 37℃,220rpm for 20h.

 

 

Oxygen exhaust

 

1. Sterilize the long needle on the nitrogen bottle by heating.

 

2. Turn on the nitrogen bottle to make sure that gas continues going out.

 

3. Connect the hydrogen production machinery to the bump until the negative pressure reaches the highest.

 

4. Keep the needle beneath the liquid layer and modulate the amount of nitrogen produced and make sure that the bubble is constantly producing. Ventilate the device by piercing the hydrogen production device.

 

5. Keep nitrogen ventilation for 5min.

 

6. Use GC to detect if there is oxygen. If there is no oxygen, use parafilm to seal the hydrogen production device. However, if there is oxygen, keep ventilating nitrogen until there is no oxygen in the device.

Quick Link

* Put your mouse on those cards to see what will happen

CONTACT

Address
  • Life Science Department
  • #163 Xianlin Blvd, Qixia District
  • Nanjing University
  • Nanjing, Jiangsu Province
  • P.R. of China
  • Zip: 210046
Email
  • nanjing_china@163.com
Social media

SPONSORS

南京大学
生命科学学院
SCHOOL OF LIFE SCIENCES
NANJING UNIVERSITY

南京大学

NANJING UNIVERSITY


南京大学
化学化工学院
SCHOOL OF CHEMISTRY AND CHEMICAL ENGINEERING
NANJING UNIVERSITY
南京大学
化学与生物医学科学研究所
INSTITUTE OF CHEMISTRY AND BIOMEDICAL SCIENCES
NANJING UNIVERSITY