Team:Nanjing-China/notebook
From 2014.igem.org
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<div class="topBarPic"><a href="https://2014.igem.org/Team:Nanjing-China/safety"><img src="https://static.igem.org/mediawiki/2014/8/84/Safety.png"/></a></div> | <div class="topBarPic"><a href="https://2014.igem.org/Team:Nanjing-China/safety"><img src="https://static.igem.org/mediawiki/2014/8/84/Safety.png"/></a></div> | ||
<div class="topBarPic" id="topPic"><a href="https://2014.igem.org/Team:Nanjing-China"><img src="https://static.igem.org/mediawiki/2014/archive/9/9a/20141015213559%21Home.png"/></a></div> | <div class="topBarPic" id="topPic"><a href="https://2014.igem.org/Team:Nanjing-China"><img src="https://static.igem.org/mediawiki/2014/archive/9/9a/20141015213559%21Home.png"/></a></div> | ||
+ | <div class="topBarPic" ><a href="https://2014.igem.org/Main_Page"><img src="https://static.igem.org/mediawiki/2014/d/d6/IGEM_official_logo.png" style="width:90px;height:70px;margin-top:-155px;margin-left:1140px"/></a></div> | ||
</div> | </div> | ||
<br><br><br><br> | <br><br><br><br> | ||
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<div class="menuItem" style="font-size:18px;"><a href="#002"><p>Protocols</p></a></div> | <div class="menuItem" style="font-size:18px;"><a href="#002"><p>Protocols</p></a></div> | ||
<div class="menuItem" style="font-size:18px;"><a href="#003"><p>Acknowledgement</p></a></div> | <div class="menuItem" style="font-size:18px;"><a href="#003"><p>Acknowledgement</p></a></div> | ||
+ | <div class="menuItem" style="font-size:18px;"><a href="#004"><p>FAQ</p></a></div> | ||
</div> | </div> | ||
<div id="contentContainer"> | <div id="contentContainer"> | ||
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<br> | <br> | ||
<div class="para"><p> | <div class="para"><p> | ||
- | <span style="font-size:24px"> | + | <span style="font-size:24px">How to make use of our bacteria?</span><br><br> |
To solve this problem, we constructed a small device like this: | To solve this problem, we constructed a small device like this: | ||
</p></div> | </p></div> | ||
Line 580: | Line 582: | ||
As is shown in the picture, if we fix the channel 1 in the end of factories’ tube, | As is shown in the picture, if we fix the channel 1 in the end of factories’ tube, | ||
after a while, open the channel 3, clean water<br> will pour out.<br><br> | after a while, open the channel 3, clean water<br> will pour out.<br><br> | ||
- | <span style="font-size:24px"> | + | <span style="font-size:24px">How to avoid engineered bacteria hazard and gene transfer?</span><br><br> |
- | Our plasmid consists 40 | + | Our plasmid consists more than 40 artificial fragments. To avoid hazard caused by engineered bacteria, |
we construct a suicide system. Bacteria with the "suicide system" will perform like this: | we construct a suicide system. Bacteria with the "suicide system" will perform like this: | ||
</p></div> | </p></div> | ||
<img src="https://static.igem.org/mediawiki/2014/5/5d/Notebook04.jpg" style="margin-left:60px"/> | <img src="https://static.igem.org/mediawiki/2014/5/5d/Notebook04.jpg" style="margin-left:60px"/> | ||
<div class="para"><p> | <div class="para"><p> | ||
- | In our lab, we will use IPTG to culture bacteria, and in the nature, bacteria will die without IPTG or Toxin. | + | In our lab, we will use IPTG to culture bacteria, and in the nature, bacteria will die without IPTG or Toxin.We proved that it works in our wet lab experiments.We also design some systems to stop horizontal gene transfer. Detail can be seen in our safety section.<br><br> |
+ | <span style="font-size:24px">How can we popularize iGEM and make people from deferent fields know synthetic biology?</span><br><br> | ||
+ | We establish iGEM club and have finished the new recruit. In the new recruit we attract many students from deferent departments who are interested in synthetic biology. In addition, we are planning to establish an information platform that students from all departments of Nanjing University can share the information related to iGEM. These activities are all supported by teachers of our university. | ||
+ | Here are some pictures in the new recruit. | ||
+ | </p></div> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/b/bc/Notebook05.jpg" style="margin-left:40px"/> | ||
+ | <div class="para"><p> | ||
+ | In addition, we also apply for a reward about environment that our university hold. | ||
</p></div> | </p></div> | ||
<br> | <br> | ||
- | <img src=" | + | <img src="https://static.igem.org/mediawiki/2014/9/97/Notebook06.jpg" style="margin-left:40px"/> |
+ | <br><br> | ||
+ | <br> | ||
+ | <img src="https://static.igem.org/mediawiki/2014/9/96/Wall.png" class="wall"/> | ||
</div> | </div> | ||
- | + | <br><br> | |
+ | <p style="text-align:center">Contact us:2014nanjingchina@gmail.com</p> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 23:05, 17 October 2014
![](https://static.igem.org/mediawiki/2014/9/96/Wall.png)
January 2014~March 2014
Summarized previous work, communicated our original ideas and plans about the project, searched papers and discussed feasibility of the projects.
Discussed the pros and cons of our 12 plans and choose one.
Designed the circuit and make out plan about experiments.
April 2014
Preparation of LB medium, plates, bacterial, antibiotics and tubes.
Transformation of Prlux, RBS, luxI, luxR, Pcons1, GFP, luxI, Ter.
Testing of chlorampenicol
Bacterial Stock, and measurement of plasmids’ concentrate.
May 2014
Preparation of plasmids ( Prlux, Plac, Ter ,luxR, Pcons).
Transformation of luxR, Pcons(21D)
Dissolution of primers and PCR of Pcons1, GFP, luxI, luxR, Prlux, Plac, Pcons2, Restriction digestion (Vector: PSBC, insert: luxI ).
Purification of digestion product, ligation and transformation
Restriction digestion of Ter fragment and Vector: luxI(PSBC), luxI(PSBA) Insert: Ter), ligation(luxI(C)-Ter, luxI(A)-Ter, (Vector: PSBA, Insert: luxI) ( Insert: Ter)
June 2014
Preparation of modeling.
Purification of PCR product, electrophoresis and restriction digestion( Ter(PSBC), Insert: luxI , luxI-T)
1th July 2014~7th July 2014
Preparation of competence cells
PCR, Restriction digestion(luxI-T, Pcons, Prlux, Ter EX, RFP EX, luxR EX, lacI EX, Plac SP, tetR XP, Pcons1 SP, lacI XP, PtetO EX, Ter ES)
Colony PCR(T-Prlux, luxR ,Ter-Prlux-RFP, Ptet, Plac, Ter, Pcons1, lacI, lysozyme, tetR, Prlux, lacI, RFP, luxR, Plac, luxR, luxI, rfp) and electrophoresis.
Preparation of plasmid(Ter, Ter-Prlux, luxR, PcI, PSBA, K12 and digestion.
Preparation of modeling.
Meeting
8th July 2014~14th July 2014
Wetlab:
Colony PCR and electrophoresis:
luxR-Ter, lacI-Ter, luxI-T, luxR-T, lacI-T, Plac-tetR, PtetO-Ter, Pcons-lacI, RFP, lacI. Purification of PCR product.
Preparation of sequencing samples. ( luxI-Ter, luxR-Ter, Plac) Restriction digestion:(Vector EX, luxR ES, luxI ES, RFP ES luxI-Ter EX, luxR-Ter EX, Plac ES, Pcons ES.)
Ligation: luxI-T, luxR-T, lacI-T, RFP-T
Transformation: luxR-Ter, luxI-Ter, lacI-Ter, RFP-Ter
Preparation of plasmids: luxI-Ter, luxR-Ter, lacI-Ter, Plac
Drylab:
Modeling
14th July 2014~21th July 2014
Wetlab:
Restriction digestion: Ter EX, luxI-Ter EX, luxR-Ter EX, luxI-Ter EX, luxR-Ter EX, Ter EX, Plac-tetR SP, Plac-tetR SP, Ter EX
Ligation:Pcons-luxR-Ter, RFP-luxR-Ter, Plac-luxI-Ter, RFP-luxI-Ter, rfp-Ter, lacI-Ter, Plac-luxI-Ter, RFP-Ter, RFP-luxI-Ter, Pcons-luxR-Ter, RFP-luxI-Ter, luxI-luxR-Ter, Pcons1-lacI-Ter
Plasmid PCR: luxR-Ter, luxI-Ter.
Electrophoresis of digestion product.
Transformation.
Drylab:
Modeling
22th July 2014~31th July 2014
Wetlab:
PCR:luxI, RFP.
Purification of digestion product.
Ligation:RFP-Ter, Plac-luxI-Ter, RFP-luxI-Ter, Pcons1-luxR-Ter, RFP-luxR-Ter, luxI-luxR-Ter, Pcons1-lacI-Ter.
Gel extraction: Ter, luxI-Ter, luxR-Ter
Preparation of plasmids: Plac-tetR, Ptet-Ter.
Drylab:
Modeling
1th August 2014~7th August 2014
Wetlab:
Plasmid PCR and electrophoresis: Pcons-lacI-Ter, Plac-luxI-Ter, RFP-luxR-Ter, luxI, lacI-Ter, PcI , Pcons-lacI-Ter, Plac-luxI-Ter, RFP-luxR-Ter.
Restriction digestion: Pcons1, Plac, Prlux, RFP, luxI, luxR-Ter, RFP-luxI-Ter., RFP-luxI-Ter EX, RFP-luxI-Ter EX, luxI-luxR- Ter EX, Prlux-RFP-Ter EX, luxR-Ter EX, Pcons-luxR-Ter ES, Pcons-luxR-Ter, Prlux-lacI-Ter.
Ligation:Pcons-lacI-Ter, Plac-luxI-Ter, Pcons-luxR-Ter, Prlux-RFP-Ter, Prlux-RFP-luxI-Ter, RFP-luxR-Ter, luxI-luxR-Ter, Pcons-lacI-Ter, Pcons-luxR-Ter, RFP-luxR-TEr, luxI-luxR-Ter, Plac-luxI-Ter, Prlux-RFP-Ter, Prlux-RFP-luxI-Ter, Prlux-RFP- luxI-Ter, PcI-lacI-Ter, Prlux-cI-Ter
Drylab:
Modeling
8th August 2014~14th August 2014
Wetlab:
Preparation of sequencing samples: RFP-luxR-Ter, Prlux-cI-Ter, PcI-lacI-Ter, Prlux-RFP-Ter
Restriction digestion: RFP-luxI-Ter, luxI-luxR-Ter, RFP-luxR-Ter, Pcons-luxR-Ter,Pcons1-luxR-Ter, Prlux-RFP-Ter
Purification: RFP-luxI-Ter, luxI-luxR-Ter, RFP-luxR-Ter
Ligation:Prlux-RFP-luxI-Ter, RFP-luxI-luxR-Ter, Prlux-RFP-luxR-Ter, RFP-luxR-Ter, RFP-luxI-luxR-Ter.
Transformation.
Drylab:
Modeling
15th August 2014~21th August 2014
Wetlab:
Preparation of plasmids:Prlux-RFP-luxR-Ter, Prlux-RFP-luxI-Ter, Prlux-RFP-luxI-luxR, Pcons-luxR-Ter, Pcons-lacI,Prlcux-cI-Ter,PcI,lacI-Ter ,
Restriction digestion: Prlux-RFP-luxI-luxR SP, Pcons-lacI SP, Prlux-lacI-Ter SP, Pcons-luxR-Ter XP, Prlux-RFP-Ter XP, Prlux-lacI-Ter-Pcons-luxR-TerSP, Pcons-lacI-Ter SP, Prlux-RFP-luxI-TerXP, Prlux-RFP-luxR-Ter XP, Prlux-RFP-luxI-luxR-Ter XP.
Ligation :Prlux-RFP-luxI-luxR-Ter, Pcons-lacI-Ter, Prlux-lacI-Ter-Pcons-luxR-Ter.
Drylab:
Modeling
22th August 2014~31th August 2014
Restriction digestion: Pcons-lacI SP.
Ligation:Pcons-lacI-Ter-Plac-luxI-Ter, Prlux-lacI-Ter-Pcons-luxR-Ter-Plac-RFP-Ter. Pcons-lacI-Ter, Prlux-RFP-luxI, Prlux-RFP-luxR, Pcons-luxR.
Preparation of plasmids:Pcons-lacI, Prlux-RFP-luxR-luxI
Continue to construct parts.
Experiments about MlrD and SDS-PAGE.
Experiments about riboswitches.
1th September 2014~15th September 2014
MlrD, HPLC and SDS-PAGE.
Experiments about riboswitches.
Preparation of plasmids:Pcons2-luxR, Plac-RFP-Ter, Prlux-RFP-Ter, Prlux-RFP, Ptet-RFP, Prlux-lacI-Ter, Ptet-RFP, Plac-luxI-Ter, luxR, PgolS-golB-PgolB
Ligation:Ptet-RFP-Ter, Prlux-RFP-luxR, Prlux-RFp-luxI, Ptet-RFP-Ter, Prlux-RFP-luxR, Prlux-RFP-luxI, Prlux-RFP-luxI, Prlux-RFP-luxR, PtetO-RFP-Ter, PtetO-RFP-Ter
Colony PCR:Ptet-RFP-Ter, Prlux-RFP-luxR, Prlux-RFP-luxI, PgolS-golB-PgolB, Pcons2-luxR
16th September 2014~30th September 2014
Preparation of plasmids:Pcons2, Pcons-luxR, PCI-lacI, Prlux-RFP-luxI-Ter, Prlux-RFP-luxR-Ter, Prlux-RFP-luxI-luxR, PgolS-golS-PgolB-tetR, PcI-lacI-Ter ,Pcons2-luxR, Prlux-RFP-luxI-luxR
Colony PCR: Prlux-RFP, Ptet-RFP-Ter, Prlux-RFP-luxI, Pcons2-luxR-Ter, Prlux-RFP-luxI-luxR-Ter, -golS-golB-tetR-Ter-PtetO-RFP-Ter-Plac-tetR-Ter, Pcons2-luxR-Ter, Prlux-RFP-luxI-luxR-Ter
Restriction digestion:luxI, luxR.
Ligation:Prlux-RFP-luxI, Prlux-RFP-luxR, Prlux-RFP, Ptet-RFP-Ter, Ptet-RFP, Pcons2-luxR-Ter-Prlux-lacI-Ter, Pcons2-lacI-Ter-Plac-luxI-Ter-Pcons2-luxR-Ter, PgolS-golS-PgolB-tetR-Ter-PtetO-RFP-Ter-Plac-tetR-Ter-Pcons2-lacI-Ter,Pgols-gols-PgolB-tetR-RFP-Ter-PtetO-RFP-Ter-Plac-tetR-Ter-Pcons2-lacI-Ter.
Finally, we finished the construction of our plasmid, at the end of September.
1th October 2014~15th October 2014
The function verification of our plasmid.
![](https://static.igem.org/mediawiki/2014/9/96/Wall.png)
1 Bacterial Transformation
Reagents and Materials: Plasmid DNA (from purified plasmid solution or ligation reaction) Competent cells (K12 or DH5α) LB agar plates (containing specific antibiotics) Liquid LB medium (without antibiotics, sterilized) Protocols: 1. Take specific competent cells out of -80℃ refrigerator, and put them on the ice. 2. Put purified Plasmid solution(5μL) or ligation product(10μL) into the competent cell solution , mix it gently and hold it on the
ice for 30 minutes. 3. Hold the mixture in 42℃ water bath for exact 90 seconds. 4. Put 900μL LB liquid medium in the mixture, put the EP tubes holding mixture in to the shaker, and shake for 1 hour at 37℃ at
proper speed. 5. Centrifuge the solution at 3000rpm for 90s. 6. Remove 860 μL solution, blend the remainder, put it on the LB plates( containing specific antibiotics and having been
activated for more than 20 minutes ) and coat the plates with sterile glass spreading rod. 7. Cultivate the bacterial for more than 10 hours. Attention: 2,4,6 are all done in super clean bench, which should be sterilized by ultraviolet for more than 15 minutes before use. 2 Colony PCR Reagents and Materials: rTag DNA polymerase( in glycerol, TaKaRa taq) 10X PCR buffer(Mg2+ free, TaKaRa) 25mM MgCl2 solution(TaKaRa) dNTP mixture(TaKaRa) ddH2O Protocols: Mixture(per reaction) 28μLddH2O 5μL 10X buffer 3μL MgCl2 1μL dNTP 1μL primer(F) 1μL primer(R) 10μL template 1μL rTaq 1. Prepare 4 EP tubes and 4 PCR tubes for each plate, put 10μL ddH2O in each EP tubes and 8μL ddH2O in each PCR tubes. 2. Pick up a little bit of colony and mix it with water in EP tubes, put 2μLof the mixture to the corresponding PCR tube with 8μL
and mix it. 3. Repeat 2 for another 3 colonies in the same plate. 4. Prepare the PCR mixture and place the tubes in the thermal cycler. 5. Set the PCR program in the thermal cycler. Stage 1(1 cycle): 95℃for 10 minutes Stage 2: 95℃ for 30 seconds, 60℃ for at least 30 seconds, 72℃ for at least 1 minutes. ( for 30 cycles. The time for 60℃ is
always half the time for 72℃, and the time for 72℃ is 1 minutes/kb ) Stage 3: 72℃ for 10 minutes Stage 4: hold at 12℃ 3 Bacterial Culture (1) Culture in liquid LB medium Reagents and Materials: Liquid LB medium ( with specific antibiotics, sterilized ) Specific antibiotics (A: Ampicillin, C: Chloramphenicol, K: Kanamycin, T: Tetracycline) Protocols: 1. Put 10ml Liquid LB medium in 50ml tubes, and add the antibiotics(antibiotics : Liquid medium = 1 : 1000) 2. Add the bacterial solution to the medium( such as the mixture of bacterial and water after picking up the colony) 3. Put the tubes into the shaker, and shake for more than 10-12 hours at 37℃ at proper speed. (2) Culture on the plate Reagents and Materials: Plate ( with specific antibiotics) Protocols: 1. Put 100-200 μL bacterial solution on the LB plates (containing specific antibiotics and having been activated for more than 20
minutes) and coat the plates with sterile glass spreading rod. 2. Cultivate the bacterial for more than 10 hours. 4 Bacterial Stock Reagents and Materials: Glycerol (50%) Protocols: 1. Put 240μL glycerol in screw cap tubes, and add 760μL overnight liquid medium. 2. Place screw cap tubes in -80℃ refrigerator. 5 Plasmid Preparation Reagents and Materials( AxyPrep Plasmid MiniPrep Kit): Buffer S1(with RNase A, stored at 4℃) Buffer S2 Buffer S3 Buffer W1 Buffer W2(with ethanol) ddH2O Protocols: 1. Put 1-4 ml overnight LB liquid medium into 2ml EP tubes, centrifuge tubes at 12000g for 1 minute and discard the
supernatant. 2. Add 250μL Buffer S1, suspend the bacterial completely. 3. Add 250μL Buffer S2, gently overturn the tubes for 4-6 times. ( This step last less than 5 minutes) 4. Add Buffer S3, gently overturn the tubes for 6-8 times, centrifuge the tubes at 12000g for 10 minutes. 5. Transfer supernatant from step 4 to Miniprep column, centrifuge the tubes at 12000g for 1 minute, discard the filtrate. 6. Place the columns back to 2ml tubes, add 500μL Buffer W1, centrifuge tubes at 12000g for 1 minute, discard the filtrate. 7. Place the columns back to 2ml tubes, add 700μL Buffer W2, centrifuge tubes at 12000g for 1 minute, discard the filtrate. 8. Repeat step 8. 9. Place columns back to tubes, centrifuge tubes at 12000g for 1 minutes. 10. Place columns to 1.5ml tubes, add 60μL 65℃ ddH2O, leave them standing at room temperature for 1 minute, and centrifuge
them at 12000g for 1 minute. 6 Restriction Digestion Reagents and Materials: Specific TaKaRa restriction endonuclease(S: SpeI, P: PstI, E: EcoRI, X: XbaI, N Nde I) 10X Specific TaKaRa buffer( When you use X and P, you should use buffer M. When you use S and P you should use buffer H.
For other situations, you can refer to the table of buffer for double digestion) Protocols: 1. Prepare the reaction mixture in PCR tubes as follows. Plasmid digestion mixture: 43μL vector 5μL specific buffer 1μL enzyme 1 1μL enzyme 2 PCR product digestion mixture: 16μL PCR product( after purification ) 2μL specific buffer 1μL enzyme 1 1μL enzyme 2 2. Place the tubes in water bath at 37℃ for specific time( The time depend on kinds of the plasmid and activity of the enzyme) 7 Ligation Reagents and Materials: T4 DNA ligase (TaKaRa) 10X T4 ligase buffer (TaKaRa) Protocols: 1. Prepare the reaction mixture in tubes. Mixture: 2μL vector 6μL insert 1μL T4 ligase buffer 1μL T4 ligase ( The volume of vector and insert depend on the concentration of them, it is better to let the mole ratio of vector: insert to be
1:10. Total volume of vector and insert should be 8μL) 2. Place the tubes in Low-temperature thermostat bath at 16℃ for more than 2 hours. 8 Agarose Electrophoresis Reagents and Materials: 1X TAE buffer REGULAR AGAROSE G-10(BIOWEST) DL2000 DNA Marker 10X Loading Buffer Protocols: 1. Prepare the agarose gel (1%). Mixture: 0.5 g REGULAR AGAROSE G-10 50ml TAE buffer Heat the mixture until boiling (about 1 minute in microwave oven). Add 5μl EB( ethidium bromide) when it is not very hot, pour it
into assembled gel pouring apparatus, wait until the gel turn solid enough. 2. Place the gel into the electrophoresis tank, and pour 1X TAE buffer until the gel is covered. 3. Add DL2000 DNA Marker and samples(with loading buffer). Run the apparatus at proper voltage (usually 120V- 135V) for
proper time (20-30 minutes). 4. Visualize the gel under ultraviolet, and record the results. Reagents and Materials: Protocols: 9 Plasmid PCR Reagents and Materials: rTag DNA polymerase( in glycerol, TaKaRa Taq ) or TaKaRa La Taq PCR buffer or 10X La Taq RCR buffer ( TaKaRa) 25mM MgCl2 solution (TaKaRa) dNTP mixture (TaKaRa) ddH2O Protocols: Mixture: 37μL ddH2O 5μL 10X buffer 3μL MgCl2 1μL dNTP 1μL primer(F) 1μL primer(R) 1μL template 1μL rTaq Polymerase 1. Prepare the mixture in PCR tubes. 2. Place the PCR tubes in the thermal cycler, and set the program. Stage 1(1 cycle): 95℃for 3 minutes Stage 2: 95℃ for 30 seconds, 60℃ for at least 30 seconds, 72℃ for at least 1 minutes. ( for 30 cycles. The time for 60℃ is
always half the time for 72℃, and the time for 72℃ is 1 minutes/kb ) Stage 3: 72℃ for 10 minutes Stage 4: hold at 12℃ 10 LB Agar Plate and LB culture medium Reagents and Materials: Tryptone(OXOID) Yeast Extract(OXOID) NaCl(Sinopharm Chemical Reagent Co..Lid) Agar Powder(Solarbio) Specific antibiotics Protocols(for 1L LB medium): (1) LB liquid cultivation Mixture: 10g Trptone 5g Yeast Extract 10g NaCl 1. Add water to the Mixture until the total volume is 1 L in beaker. 2. Place the beaker on the magnetic stirrers, and stir it until solid resolves. 3. Sterilize the medium in high-handed sterilization pan at program P7. (the container should be opened partly) (2) Plate Mixture: 10g Trptone 5g Yeast Extract 10g NaCl 1. Add water to the Mixture until the total volume is 1 L in beaker. 2. Place the beaker on the magnetic stirrers, and stir it until solid resolves. Pour the solution into a bottle containing 15g Agar
Powder. 3. Sterilize the medium in high-handed sterilization pan at program P7. (the container should be opened partly) 4. Add specific antibiotics when it is not very hot and shake it. 5. Pour the LB medium to the plate (25ml/each) , wait until it solidify (done in the super clean bench) and place plates to 4℃
refrigerator. 11 Primer dissolution Reagents and Materials: ddH2O Protocols: 1. Shortly centrifuge the EP tubes containing primer powder. 2. Slowly open the tubes, add ddH2O until the concentrate of primer is 20 mM, vortex them, put the tubes in -30℃
refrigerator. 12 Making competent cells Reagents and Materials: CaCl2 and glycerol mixture(0.1mol/L CaCl2), CaCl2-MgCl2(80mmol/LMgCl2, 20mmol/L CaCl2) K12 or DH5α plate Protocols: 1. Pick a colony from the plate and put it to 100 mL LB medium, and cultivate the medium. 2. Measure OD600, until OD600 is 0.35. 3. Precool two 50mL tubes, 80 EP tubes, CaCl2 and glycerol mixture, CaCl2-MgCl2 4. Put 100ml bacterial solution to two 50ml tubes, and place them on ice for 10 minutes. 5. Centrifuge tubes at 4100rpm for 10 minutes, at 4℃. 6. Discard the supernate. Put the tubes upside down without caps for 1 minute to get rid of the supernate remained. 7. Resuspend the bacterial by 30ml cold 0.1mol/L CaCl2-MgCl2 per 50ml medium at the beginning. 8. Centrifuge at 4100rpm, 4℃ for 10 minutes 9. Discard the supernate. Put the tubes upside down without caps for 1 minute to get rid of the supernate remained. 10. Resuspend the bacterial by 2ml cold 0.1mol/L CaCl2 per 50ml medium at the beginning. 11. Place 50μL mixture in each tube, and place them in -80℃ refrigerator. 13 Gel Extraction Reagents and Materials: Buffer DE-A Buffer DE-B Buffer W1 Buffer W2(with alcohol) Isopropanol ddH2O Protocols: 1. Cut wanted bands on the gel place them in the tubes and calculate their volumes. 2. Pour triple volume of Buffer DE-A in the tubes, keep tubes at 75℃ until the gel melt completely. 3. Add 1.5 times volume of Buffer DE-B, blend it.( If the fragment you want is shorter than 400bp, one volume of isopropanol
need to be added) 4. Add mixture from step 3 to the columns, centrifuge them at 12000g for 1 minute and discard the filtrate. 5. Add 500μL Buffer W1, centrifuge them at 12000g for 30 seconds and discard the filtrate. 6. Add 700μL Buffer W2, centrifuge them at 12000g for 30 seconds, and discard the filtrate. 7. Repeat step 6. 8. Centrifuge the tubes at 12000g for 1 minute. 9. Place columns to 1.5ml tubes, add 60μL 65℃ ddH2O, leave them standing at room temperature for 1 minute, and centrifuge
them at 12000g for 1 minute. 14 Purification of PCR product and Restriction Digestion product Reagents and Materials: Buffer PCR-A Buffer W2 ddH2O Protocols: 1. Add three volume of Buffer PCR-A to the mixture (if Buffer PCR-A needed less than 100μL, add 100μL ), centrifuge them at
12000g for 1 minute and discard filtrate. 2. Add 700μL Buffer W2, centrifuge at 12000g for 1 minute, and discard the filtrate. 3. Add 400μL Buffer W2, centrifuge at 12000g for 1 minute, and discard the filtrate. 4. Place columns to 1.5ml tubes, add 60μL 65℃ ddH2O, leave them standing at room temperature for 1 minute, and centrifuge
them for 1 minute. 15 Process with AP enzyme Regents and Materials: AP enzyme ( Alkaline Phasthatase) Buffer( 10XBS buffer W : ddH2O = 6 : 3) Protocols: Mixture: 50μL digested product 9μL Buffer 1μL AP enzyme 1.blend the mixture, and put it in Low-temperature thermostat bath at 25℃ for 30 minutes.
![](https://static.igem.org/mediawiki/2014/9/96/Wall.png)
Here we express our gratitude to technical support from Prof. Zhao Jing’s lab, Nanjing University. We express our gratitude to the great support from Professor Kong Lingdong and Professor Yang Sihai, and the help from
other teachers from the School of Life Sciences, Nanjing University. And especially, we express our heartfelt thanks to: 2014-Tsinghua-A, for their help in our model building. 2014-Jilin-China, for their help with the mrlA gene synthesis. 2014-NCTU_Formosa, for their great seminar. Wang Jiangning, for his help in buliding the wiki. Finally, we express great thanks to 2014-NJU-QIBEBT and 2013-Nanjing-China for providing us with some important substance,
![](https://static.igem.org/mediawiki/2014/9/96/Wall.png)
How to make use of our bacteria?
To solve this problem, we constructed a small device like this:
![](https://static.igem.org/mediawiki/2014/c/c6/Notebook02.jpg)
a normal injector with a filtering membrane front. We will culture our bacteria on the filtering
membrane anteriorly. When
used, you just need to take in some dirty water and eject them,
then you will get clean water.
Moreover, we try to design a device to clean the water discharge from factories.
![](https://static.igem.org/mediawiki/2014/9/9d/Notebook03.jpg)
As is shown in the picture, if we fix the channel 1 in the end of factories’ tube,
after a while, open the channel 3, clean water
will pour out.
How to avoid engineered bacteria hazard and gene transfer?
Our plasmid consists more than 40 artificial fragments. To avoid hazard caused by engineered bacteria,
we construct a suicide system. Bacteria with the "suicide system" will perform like this:
![](https://static.igem.org/mediawiki/2014/5/5d/Notebook04.jpg)
In our lab, we will use IPTG to culture bacteria, and in the nature, bacteria will die without IPTG or Toxin.We proved that it works in our wet lab experiments.We also design some systems to stop horizontal gene transfer. Detail can be seen in our safety section.
How can we popularize iGEM and make people from deferent fields know synthetic biology?
We establish iGEM club and have finished the new recruit. In the new recruit we attract many students from deferent departments who are interested in synthetic biology. In addition, we are planning to establish an information platform that students from all departments of Nanjing University can share the information related to iGEM. These activities are all supported by teachers of our university.
Here are some pictures in the new recruit.
![](https://static.igem.org/mediawiki/2014/b/bc/Notebook05.jpg)
In addition, we also apply for a reward about environment that our university hold.
![](https://static.igem.org/mediawiki/2014/9/97/Notebook06.jpg)
![](https://static.igem.org/mediawiki/2014/9/96/Wall.png)
Contact us:2014nanjingchina@gmail.com