Team:Shenzhen/Result/YAO.Suicider
From 2012.igem.org
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<ul><p>DLD3 promoter + Holin +ADH terminator</p></ul> | <ul><p>DLD3 promoter + Holin +ADH terminator</p></ul> | ||
</div> | </div> | ||
- | + | <div class="context"> | |
+ | <h5>Identification of T7 RNAP Function in Nucleus</h5> | ||
+ | <ul><li>I. Construction of Vector </li></ul> | ||
+ | <ul><p>1. Bacterial culture (BBa_I712074(T7 poromter,Amp resistant or Kana resistant),E0840(GFP generator,Amp resistant)</p> </ul> | ||
+ | <ul><p>2. Plasmid extraction for BBa_I712074,E0840( Tiangen normal plasmid extraction kits</p> </ul> | ||
+ | <ul><p>3. Measure the concentration of plasmid by NANODROP 2000(Thermo)</p> </ul> | ||
+ | <ul><p>4. Digestion: (enzymes are all from Takara </p> | ||
+ | <p> BBa_I712074 2-3ug</p> | ||
+ | <p> PstI 2ul</p> | ||
+ | <p> SpeI 2ul</p> | ||
+ | <p> 10 x H Buffer 5ul</p> | ||
+ | <p> ddH2O 50ul</p> | ||
+ | <p> E0840 2-3ug</p> | ||
+ | <p> XbaI 2ul</p> | ||
+ | <p> PstI 2ul</p> | ||
+ | <p> 10 x H Buffer 5ul</p> | ||
+ | <p> ddH2O 50ul</p> | ||
+ | <p> 37oC 3h</p> | ||
+ | <p> 80 oC 20min</p></ul> | ||
+ | <ul><p>5. Electrophoresis: </p> | ||
+ | <p> 1% agarose gel, 120V, after 45min,EB dye. </p> </ul> | ||
+ | <ul><p>6. Gel recovery of GAL(EcoRI/SpeI), Holin(XbaI/PstI) (QIAquick Gel recovery kits)</p> </ul> | ||
+ | <ul><p>7. Measure the concentration of fragment of interest by NANODROP 2000(Thermo)</p> </ul> | ||
+ | <ul><p>8. Linage: </p> | ||
+ | <p> BBa_I712074 ((stI/SpeI) 2ul</p> | ||
+ | <p> E0840 (PstI/XbaI) 6ul</p> | ||
+ | <p> T4 ligase 1ul</p> | ||
+ | <p> T4 ligase Buffer 1ul</p> | ||
+ | <p> 16 oC 3 hours</p> </ul> | ||
+ | <ul><p>9. Transduction: </p> | ||
+ | <p> 1. UV sterilization half an hour before, get DH5a (competent yeast) out of fridge, thawing in water bath. </p> | ||
+ | <p> 2. 10μlinkage production with 50μl competent yeast cells, ice-bath for 30mins. </p> | ||
+ | <p> 3. 42oC ice-bath heat shock for 50s. </p> | ||
+ | <p> 4. Stewing on ice for 2mins. </p> | ||
+ | <p> 5. Add 500μl LB medium without antibiotic, 37℃ shaking(225rpm/min), recover it for culturing it for 1 hour. </p> | ||
+ | <p> 6. 3000rpm/min for 5mins, abandon the supermate, mixing the left. </p> | ||
+ | <p> 7. 60μl broth spread the Amp anti-LB soli<UL> | ||
+ | <LI>March 12th, 2012 </li> | ||
+ | <P>Events:</P> | ||
+ | <P>March 12th. The first iGEM meeting was a very serious one, and there were only 5 members: Kang Chen Zhang Ou and Gong ,respectively.</P> | ||
+ | <P>We decided to build a list about the whole previous iGEM projects and divided it into five parts: America for Kang and Chen, Europe for Zhang and Ou, Asia for Gong. All of us were in charge of scanning the projects, summarized them and gave the other members a brief description. Each one for one minute.</P> | ||
+ | </UL> | ||
+ | <UL> | ||
+ | <LI>March 15th – April 13th, 2012</li> | ||
+ | <P>Events:</P> | ||
+ | <P>Though a recruitment talk with the other classmate in BGI, nearly 20 students and two instructors became members of us. We established a team consisted of eight schools—SCUT, SCNU, SCU, UESTC, WHU, HUST, SEU, UST.</P> | ||
+ | <P>We continued to sum up the projects from 2008 to 2011, and in the meanwhile, we separated the whole into more parts and assigned to the other members.</P> | ||
+ | <P>Finally, we finished the summary. </P> | ||
+ | <P>And we also discussed what we should do, we originally put up with 4 schemes: Schrodinger’s cat—randomly producing 0 and 1; Density sensing application--based on the result of JIandong Wang’s research; Christmas tree—sparks different colors ceaselessly; Yeast artificial organelle—transform mitochondria into a biofactory. The former 3 projects were gave up due to the similarity to the previous iGEM projects. We lay down our consideration on Yeast artificial organelle.</P> | ||
+ | <P>Chen Yu search more articles about mitochondria transgene and mitochondrial signaling pathways he can and pack it as a file of endnote.</P> | ||
+ | <P>We clarify our projects as a combination of five modules: NAD+/NADH Sensor, self-killer, post-invader, sel-control transporter and artemisinin producer.</P> | ||
+ | <P>We divided our group into five parts according to the modules and select two members of each modules to make up two new squads—modeling construction and lab experiments.</P> | ||
+ | </UL> | ||
+ | 2012/3/7 | ||
+ | MISSION START | ||
+ | 1. | ||
+ | TEAM BUILDING: OICQ GROUP; | ||
+ | 2. | ||
+ | ARRANGEMENTS FOR THE FIRST SEMINAR. | ||
+ | 2012/3/8 noon | ||
+ | THE FIRST SEMINAR: | ||
+ | 1. | ||
+ | Discussion about our project; | ||
+ | 2. | ||
+ | Registration for iGEM 2012 and self-introduce. | ||
+ | 2012/3/14 | ||
+ | THE SECOND SEMINAR: | ||
+ | 1. | ||
+ | Discussion about our project; | ||
+ | 2. | ||
+ | Task assignment for latter seminar mainly about previous teams’ projects; | ||
+ | 3. | ||
+ | New member’s introduction. | ||
+ | 2012/3/15 | ||
+ | Group email application succeeded. | ||
+ | 2012/3/17 | ||
+ | Quan ZHOU withdrew from the team. | ||
+ | 2012/3/18 | ||
+ | Jianhui GONG made a list of all projects of iGEM 2008-2011 and assigned each one’s seminar | ||
+ | task. | ||
+ | 2012/3/19 | ||
+ | THE THIRD SEMINAR: | ||
+ | 1. | ||
+ | Introduction of new members; | ||
+ | 2. | ||
+ | Rearrangements for everyone’s task. | ||
+ | 2012/3/22 | ||
+ | THE FOURTH SEMINAR: Introduction of a few projects of iGEM 2008-2011. | ||
+ | 2012/3/26 | ||
+ | THE FIFTH SEMINAR: Introduction of a few projects of iGEM 2008-2011. | ||
+ | 2012/3/29 | ||
+ | THE SIXTH SEMINAR: | ||
+ | 1. | ||
+ | Introduction of a few projects of iGEM 2008-2011; | ||
+ | 2. | ||
+ | Introduction of new members; | ||
+ | 3. | ||
+ | Rearrangements for everyone’s task again. | ||
+ | 2012/4/5 | ||
+ | THE SEVENTH SEMINAR: Introduction of a few projects of iGEM 2008-2011. | ||
+ | 2012/4/7 | ||
+ | TEAM BUILDING ACTIVITY: KTV and dine outside together. | ||
+ | 2012/4/10 | ||
+ | THE EIGTHTH SEMINAR: Each modules and logo designed.</ul></div>d plat, 37℃ inversed culture in incubation overnight. </p> </ul> | ||
+ | <ul><p>10. Colony PCR: </p> | ||
+ | <p> dNTPmix(2.5mM) 1μl</p> | ||
+ | <p> PCR 10×buffer2μl</p> | ||
+ | <p> VF 0.5μl</p> | ||
+ | <p> VR 0.5μl</p> | ||
+ | <p> Ex Taq 0.5μl</p> | ||
+ | <p> H2O 15.5μl</p> | ||
+ | <p> Primer</p> | ||
+ | <p> VF: TGCCACCTGACGTCTAAGAA</p> | ||
+ | <p> VR: ATTACCGCCTTTGAGTGAGC</p> | ||
+ | <p> 94℃ 3min</p> | ||
+ | <p> 94℃ 30s</p> | ||
+ | <p> 55℃ 30s </p> | ||
+ | <p> 72℃ 1min</p> | ||
+ | <p> 72℃ 10min</p> | ||
+ | <p> 12℃ ∞</p> | ||
+ | <p> 30 cycles</p> </ul> | ||
+ | <ul><p>11. 1%~1.5% agarose gel electrophoresis, <130V, 40mins. </p> </ul> | ||
+ | <ul><p>12. Sequencing: </p> | ||
+ | <p> Pick the positive colony(colony PCR verification), marking lines and culture(Cmr resistant), sequencing. Name the right vector PSB1AK3-T7 poromter+GFP+T7 terminater</p> | ||
+ | </ul> | ||
+ | <ul><li>II. Construction of Yeast Expression Vector</li></ul> | ||
+ | <ul><p>1. Culture of PSB1AK3-T7 poromter+GFP+T7 terminater (Cmr resistant)gz-YEP352(AmpResistant, we have replaced the URA mark with trp mark)</p></ul> | ||
+ | <ul><p>2. Plasmid extraction: PSB1AK3-T7 poromter+GFP+T7 terminater,gz-YEP352</p> </ul> | ||
+ | <ul><p>3. Digestion: </p> | ||
+ | <p> Gz-YEP352 2ug </p> | ||
+ | <p> XbaI 2ul</p> | ||
+ | <p> PstI 2ul</p> | ||
+ | <p> 10 x H Buffer 5ul</p> | ||
+ | <p> ddH2O 50ul</p> | ||
+ | <p> PSB1AK3-T7 poromter+GFP+T7 terminater 2ug </p> | ||
+ | <p> XbaI 2ul </p> | ||
+ | <p> Pst 2ul</p> | ||
+ | <p> 10 x H Buffer 5ul </p> | ||
+ | <p> ddH2 50ul</p> </ul> | ||
+ | <ul><p>4. Electrophoresis: </p> | ||
+ | <p> 1% agarose gel, 120V, after 45min,EB dye. </p> </ul> | ||
+ | <ul><p>5. Gel recovery of gz-YEP352 (XbaI /PstI)PSB1AK3-T7 poromter+GFP+T7 terminater (XbaI / PstI) (Qiagen Gel recovery kits) </p> </ul> | ||
+ | <ul><p>6. Lingake: </p> | ||
+ | <p> Gz-YEP352 (XbaI /PstI) 100ng</p> | ||
+ | <p> PSB1AK3-T7 poromter+GFP+T7 terminater 100ng</p> | ||
+ | <p> T4 buffer 1ul</p> | ||
+ | <p> T4 ligase 1ul</p> | ||
+ | <p> ddH2O 10ul</p> | ||
+ | <p> 16oC overnight</p> </ul> | ||
+ | <ul><p>7. Transduction: </p> | ||
+ | <p> 1. UV sterilization half an hour before, get DH5a (competent yeast) out of fridge, thawing in water bath. </p> | ||
+ | <p> 2. 10μlinkage production with 50μl competent yeast cells, ice-bath for 30mins. </p> | ||
+ | <p> 3. 42oC ice-bath heat shock for 50s. </p> | ||
+ | <p> 4. Stewing on ice for 2mins. </p> | ||
+ | <p> 5. Add 500μl LB medium without antibiotic, 37℃ shaking(225rpm/min), recover it for culturing it for 1 hour. </p> | ||
+ | <p> 6. 3000rpm/min for 5mins, abandon the supermate, mixing the left. </p> | ||
+ | <p> 7. 60μl broth spread the Amp anti-LB solid plat, 37℃ inversed culture in incubation overnight. </p> </ul> | ||
+ | <ul><p>8. Clony PCR: | ||
+ | <p> dNTPmix(2.5mM) 1μl</p> | ||
+ | <p> PCR 10×buffer 2μl</p> | ||
+ | <p> M13_pUC_fwd_primer 0.5μl</p> | ||
+ | <p> M13_reverse_primer 0.5μl</p> | ||
+ | <p> Ex Taq 0.5μl</p> | ||
+ | <p> H2O 15.5μl</p> | ||
+ | <p> 94℃ 3min</p> | ||
+ | <p> 94℃ 30s</p> | ||
+ | <p> 55℃ 30s </p> | ||
+ | <p> 72℃ 1.5min</p> | ||
+ | <p> 72℃ 10min</p> | ||
+ | <p> 12℃ ∞</p> | ||
+ | <p> 30 cycles</p> </ul> | ||
+ | <ul><p>9. 1.5% agarose gel electrophoresis, <130V, 40mins. </p> </ul> | ||
+ | <ul><p>10. Sequencing: </p> | ||
+ | <p> Pick the positive colony(colony PCR verification), marking lines and culture(Cmr resistant), sequencing. Name the right vector:EP352-GAL1,Holin-ADH1</p> | ||
+ | </ul> | ||
+ | <ul><li>III. Yeast Transformation Experiment:</li></ul> | ||
+ | <ul><p>1. plasmid extraction. </p> </ul> | ||
+ | <ul><p>2. Preparation of Competent Yeast Cells - LiAc Method</p> | ||
+ | <p> 1.treak a YPDA agar plate with a small portion of AH109 or Y187. Incubate the plate upside down at 30°C until colonies appear(~3days). Yeast strains can be stored for up to 1 month at 4°C on YPDA medium in culture plates. </p> | ||
+ | <p> 2.Prepare 1.1X TE/LiAc Solution </p> | ||
+ | <p> 3.Prepare YPDA liquid medium (Yeast Protocols Handbook) </p> | ||
+ | <p> 4.Inoculate one colony(<4 weeks old, 2–3mm in diameter) into 3ml of YPDA medium in a sterile, 15-ml centrifuge tube. </p> | ||
+ | <p> 5.Incubate at 30°C with shaking for 8hr. </p> | ||
+ | <p> 6.Transfer 5µl of the culture to a 250-ml flask containing 50ml of YPDA. </p> | ||
+ | <p> 7.Incubate at 30°C with shaking at 230–250 rpm for 16–20hr.The OD600 should reach 0.15–0.3. </p> | ||
+ | <p> 8.Centrifuge the cells at 700 x g for 5min at room temperature. </p> | ||
+ | <p> 9.Discard the supernatant and resuspend the cell pellet in 100 ml of YPDA. </p> | ||
+ | <p> 10.Incubate at 30°C for 3–5hr (OD600= 0.4–0.5). </p> | ||
+ | <p> 11.Centrifuge the cells at 700 x g for 5min at room temperature. </p> | ||
+ | <p> 12.Discard the supernatant and resuspend the cell pellet in 60ml of sterile, deionized H2O. </p> | ||
+ | <p> 13.Centrifuge the cells at 700 x g for 5min at room temperature. </p> | ||
+ | <p> 14.Discard the supernatant and resuspend the cells in 3 ml of 1.1 X TE/LiAc Solution. </p> | ||
+ | <p> 15.Split the resuspension between two 1.5-ml microcentrifuge tubes (1.5 ml per tube). </p> | ||
+ | <p> 16.Centrifuge each tube at high speed for 15 sec. </p> | ||
+ | <p> 17.Discard the supernatant and resuspend each pellet in 600 µl of 1.1 X TE/LiAc Solution. </p> </ul> | ||
+ | <ul><p>3.Transformation: </p> | ||
+ | <p> 1. Gz-YEP352-T7poromter+GFP+T7terminater 0.2ug </p> | ||
+ | <p> Sperm DNA*(10 mg/ml) 0.1mg</p> | ||
+ | <p> Sperm DNA :when prepared, water-bath boiling for 20mins, then ice-bath, -20°C preservation. </p> | ||
+ | <p> 2. 100 ul dense medium of yeast competent cell by 1×TE/LiAc. Whirlpool oscillation. </p> | ||
+ | <p> 3. 600 ulPEG/LiAc, oscillation strongly, 30°C,200rpm, culture oscillating for 30mins. </p> | ||
+ | <p> 4. Add 70ul DMSO, mixing it slightly and 42oC heat shock by water-bath for 15mins, ice-bath for 1-2mins</p> | ||
+ | <p> 5. Centrifuging for 14,000rpm for 5sec, abandon the supermate, 0.1ml 1x TE suspense the cell precipitation, spread it to SD/ -Ura-trp solid plat, 30°C culture reversely for 3 days. </p> </ul> | ||
+ | <ul><p>6. Holin function identification: </p> | ||
+ | <p> Pick a SD/ -Ura-trp-cultured colony and inoculate it to 50ml YPDA medium, 30°C,220rpm culture reelingly to OD-0.1, transfer 20ml to 50ml sterile medium (alpha-galactose), versus is added by equivalent water. Measuring the OD value one time for one hour and drawing the curve. </p> | ||
+ | <p> Result analyzation: </p> | ||
+ | <p> If the bacteria in alpha-galactose show flurorescence, that shows T7 RNAP/T7 promoter can work. </p> | ||
+ | </ul> | ||
+ | </div> | ||
<div class="context"> | <div class="context"> | ||
<h5>Protocol: Test of Holin Function</h5> | <h5>Protocol: Test of Holin Function</h5> |
Revision as of 10:53, 26 September 2012