Team:Nanjing-China/Notebook
Notebook
March
We established Nanjing-China 2019 in March this year. Both the team leader and all team members are new iGEMers, in which case great challenges were ahead of us. We determined that we work on gas sensors and then read a lot of papers and conceived the outline of our project.
April
In April, we started synthesizing primers and genes with the help of our PI weiwei and instructor Xin Wang. We also made a proposal within School of Life Science to secure funding and support.
May to August
From May to August, we were busy with human practice and experiments. At the end of August, we sent 8 students to attend CCIC in Shenzhen and met up with iGEMers from all over China. We made a presentation about our project so that we got to know what they thought about our project.
September and October
In September and October, we visited SEU in Nanjing to discuss our model, and we also formed collaboration with team Fudan-TSI. We also spread no effort in building wiki and modeling before November. We had been making beautiful original design throughout the whole time. We were so lucky to have talented and hard-working students to get everything neatly done before the Giant Jamboree.
Protocol
AxyPrep Plasmid Miniprep Spin Protocol
1. Collect 1-4 ml of overnight LB culture. Centrifuge at 12,000×g for 1 minute to pellet the bacteria. Decant or pipette off as much of the supernatant as practical. Note: When using rich broths such as LBG or 2×YT, reduce the culture volume by half. Excessive bacteria will reduce lysis efficiency, resulting in low yield and reduced purity of the plasmid DNA. Do not exceed 2 ml of bacterial culture grown in rich broth. 2. Resuspend the bacterial pellet in 250 μl of Buffer S1 by vortexing. Please be sure that the bacteria are completely resuspended before proceeding. Note: Be sure that RNase A has been added into Buffer S1. 3. Add 250 μl of Buffer S2, and mix by gently inverting the tube for 4-6×. Do not vortex. Note: Vigorous shaking or vortexing will cause shearing of the bacterial genomic DNA and result in the contamination of the plasmid DNA. Note: After use, the buffer S2 bottle should be closed immediately in order to avoid neutralization of NaOH by ambient CO2. Note: Buffer S3 (Step 4, below) must be added within 5 minutes. 4. Add 350 µl of Buffer S3, and mix by gently inverting 6-8×. Centrifuge at 12,000×g for 10 minutes to clarify the lysate. Do not vortex. Note: Vigorous shaking or vortexing will result in contamination with genomic DNA. 5. Place a Miniprep column into an uncapped 2 ml Microfuge tube (provided). Transfer the clarified supernatant from Step 4 into the Miniprep column. Transfer the Miniprep column and 2 ml Microfuge tube to microcentrifuge and spin at 12,000×g for 1 minute. 6. Optional step: Buffer W1 Wash Washing with Buffer W1 is required only in cases where the plasmid has been propagated in an endA+ bacterial strain. These strains often exhibit high levels of endonuclease activity which will degrade the plasmid DNA. Proceed to Step 7 if an endA- bacterial strain is used. Pipette 500 µl of Buffer W1 into each Miniprep column. Centrifuge at 12,000×g for 1 minute. 7. Pipette 700 µl of Buffer W2 into each Miniprep column. Centrifuge at 12,000×g for 1 minute. Note: Make sure that the volume of ethanol specified on the bottle label has been added to the Buffer W2 concentrate 8. Optional Step: Discard the filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Add 700 μl of Buffer W2 to the Miniprep column and centrifuge at 12,000×g for 1 minute. Note: Two washes with Buffer W2 are used to ensure the complete removal of salt, eliminating potential problems in subsequent enzymatic reactions. 9. Discard filtrate from the 2 ml Microfuge tube. Place the Miniprep column back into the 2 ml Microfuge tube. Centrifuge at 12,000×g for 1 minute. 10. Transfer the Miniprep column into a clean 1.5 ml Microfuge tube (provided). To elute the purified plasmid DNA, add 60~80 µl of Eluent (or deionized water) to the center of the membrane. Let it stand for 1 min at room temperature. Centrifuge at 12,000×g for 1 minute..
Agarose Gel Electrophoresis
1.Pouring a Standard 1% Agarose Gel:
1.1Measure 1 g of agarose.
1.2Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
1.3 Microwave for 1-3 min until the agarose is completely dissolved (Microwave for 30-45 sec, stop and
swirl, and then continue towards a boil)
1.4 Let agarose solution cool down to about 50 °C (about when you can comfortably keep your hand on
the flask), about 5 mins.
1.5 Add dye to a final concentration of 1/10000.
1.6 Pour the agarose into a gel tray with the well comb in place.
1.7 let the gel sit at room temperature for 20-30 mins, until it has completely solidified.
2.Loading Samples and Running an Agarose Gel:
2.1 Add loading buffer to each of DNA samples.
2.2 Once solidified, place the agarose gel into the gel box (electrophoresis unit).
2.3 Fill gel box with 1xTAE until the gel is covered.
2.4 Carefully load a molecular weight ladder into the first lane of the gel.
2.5 Carefully load your samples into the additional wells of the gel.
2.6 Run the gel at 120 V until the dye line is approximately 75-80% of the way down the gel.A typical
run time is about 0.5 hours.
2.7 Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel
from the gel box.
2.8 Using any device that has UV light, visualize DNA fragments.
PCR amplification
Takara PrimerSTAR Max DNA Polymerase
PCR Reaction solution composition（50 μl system）:
Volume Final Conc.
2×PrimeSTAR GC Buffer (Mg2+ plus) 25 μl 1×
dNTP Mixture (2.5 mM each) 4 μ l 200 μM each
Primer 1 10 - 15 pmol 0.2 - 0.3 μM
Primer 2 10 - 15 pmol 0.2 - 0.3 μM
Template ＜200 ng
PrimeSTAR HS DNA Polymerase (2.5 U/μl) 0.5 μl 1.25 U/50 μl
Sterile water up to 50 μl
PCR clean-up
AxyPrep PCR clean-up kit: 1. Add 3 volumes of PCR-A to 1 volume of the PCR sample and mix. 2. Place a spin column in a provided collection tube. Apply the sample to the column and centrifuge for 1min. 3. Discard flow-through and place the column back into the same tube. 4. Add 0.7 ml Buffer W2 to the column and centrifuge for 30–60 s. 5. Discard flow-through and place the column back in the same tube. 6. Add 0.4 ml Buffer W2 to the column and centrifuge for 30–60 s. 7. Place the column in a clean 1.5 ml microcentrifuge tube. 8. To elute DNA, add 30 µl water (pH 7.0–8.5) to the center of the membrane, let the column stand for 1 min, and centrifuge the column for 1 min.
Restriction enzyme digestion
System：10ul/50ul
10ul system：
DNA/plasmid： 8ul
Buffer： 1ul
Enzyme1： 0.5ul
Enzyme2： 0.5ul
Reaction temperature: 37℃
Reaction time was determined by enzyme activity and sample amount.
Ligation
10ul system:
Insert calculate
Vector calculate
T4 DNA ligase 0.5ul
10×buffer 1ul
Water up to 10 μl
Control a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
Gently mix the reaction by pipetting up and down and microfuge briefly.
Incubate at 16°C for 30 minutes.
Transformation Protocol Using Heat Shock
1. Take competent E.coli cells from –80℃ freezer. Keep tubes on ice. 2. Turn on water bath to 42℃ 3. Add 10ul of circular DNA into E.coli cells. Incubate on ice for 30 min. 4. Put tube(s) with DNA and E.coli into water bath at 42℃ for 80 seconds. 5. Put tubes back on ice for 2 minutes to reduce damage to the E.coli cells. 6. Add 1 ml of SOC medium. Incubate tubes for 1 hour at 37℃ with shaking at 200rpm. 7. Spread about 100 ul of the resulting culture on LB plates (with appropriate antibiotic added – usually Ampicillin or Kanamycin.) Grow overnight. 8. Pick colonies about 12-16 hours later.
Competent DH5alpha cell preparation
Day1: 1. Flame the metal inoculating loop until it is red got and then cools it down. 2. Scrape off a portion from the top of the frozen glycerol stock. 3. Streak it onto the LB plate. 4. Put the stock back to -80℃ immediately. 5. Leave the plates for 5 minutes and place them upside down in the 37℃ incubator for 16-20 hours. Day2 6. Pick a single colony into 5ml of LB medium. 7. inoculate the culture overnight at 37℃ with shaking at 200rpm. Day3 8. Inoculate 100ml LB medium with 1ml of saturated overnight culture. 9. Shake at 37℃ until OD600=0.5 (usually 2-3 hours). 10. Place in an ice bath for 10 minutes. 11. Pre-cool solution, centrifuge, pipette tips, falcon, Eppendorf. 12. Transfer the culture into two pre-chilled 50ml tubes. 13. Centrifuge at 4000rpm for 10 minutes at 4℃. 14. Remove the medium, resuspend the cell pellet with 30ml ice-cold 100mM CaCl2-MgCl2 by swirling on ice gently. 15. Incubate on ice for 10 minutes. 16. Centrifuge at 4000rpm for 10 minutes at 4℃. 17. Remove the medium, resuspend the cell pellet with 2ml ice-cold 100mM CaCl2- glycerol by swirling on ice gently. 18. Freeze 100ul aliquots and store in -80℃.
Activate the strains
1. Flame the metal inoculating loop until it is red got and then cools it down.
2. Scrape off a portion from the top of the frozen glycerol stock.
3. Streak it onto the LB plate.
4. Put the stock back to -80℃ immediately.
5. Leave the plates for 5 minutes and place them upside down in the 37℃ incubator for 8-10 hours.
Cultivate in LB
1. Pick a single colony into 5ml/50ml of LB medium (with appropriate antibiotic added).
2. Inoculate the culture overnight at 37℃ or for 8-10 hours with shaking at 200rpm.
Cultivate in Synthetic municipal wastewater (SMW)
1. Inoculate the culture overnight at 37℃ or for 8-10 hours with shaking at 200rpm. 2. Calculate the culture beforehand to attain an initial OD600 of 0.15 in SMW. 3. Centrifuge at 6000rpm for 5 minutes, and wash once with deionized water. 4. Resuspend the cell pellet with 5ml SMW, transfer them all to the 100ml SMV, Incubating for 10-14 hours at 37℃ with shaking at 200rpm. 5. Monitor the OD600 and the pi concentration each hour, until the bacteria cannot grow anymore or cannot remove the pi anymore.
Media component
A.LB medium：
Tryptone 10g/L
Yeast extract 5 g/L
NaCl 10g/L
Deionized water up to 1 L
* adjust pH： 1M NaOH 1ml
B.Synthetic municipal wastewater (SMW)
Glucose 0.3g/L
Sodium acetate 0.15g/L
Tryptone 0.1g/L
Yeast extract 0.01g/L
K2HPO4 ·3H2O 0.1472g/L
MgSO4 ·7H2O 0.262g/L
NH4Cl 0.18g/L
NaCl 0.05g/L