Team:SEU A/Experiment/proof4

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<h1>The confirmation experiment of Conjugation</h1>
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<h1>Death gene</h1>
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<span>Experiment Notebook.</span>
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<span>The confirmatory experiment of Death Gene</span>
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       <a href="https://2012.igem.org/Team:SEU_A/Experiment/proof4"><img src='https://static.igem.org/mediawiki/2012/8/8a/Seua_exp_death.png' width='200' height='200'></img></a>
       <a href="https://2012.igem.org/Team:SEU_A/Experiment/proof4"><img src='https://static.igem.org/mediawiki/2012/8/8a/Seua_exp_death.png' width='200' height='200'></img></a>
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       <h2>The confirmation experiment of Sweet gene</h2></br>
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       <h2>The confirmation experiment of Death Gene</h2></br>
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</html>[[Media:The confirmatory experiment of Death gene.pdf|The confirmatory experiment of Death gene.pdf]]<html></br>
      
      
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       Because this part contains oriT, the R plasmid nic region is where the relaxosome nicks the plasmid and conjugative transfer by R plasmid machinery begins. The function of this kit (K873003) is: when there is a RP4 plasmid, conjugation will be induced to start. We use BL21 which carry the K873003 plasmid as donor bacteria while DH5 α acts as recipient bacteria, and the helper is the HB101 strain who carry the RP4 plasmid. According to the document, DH5 α anti Nalidixic Acid HB101 has resistance over Ampicillin and Kanamycin, and BL21 has Chloramphenicol resistance. If the conjugation happens, the recipient bacterium will not only anti Nalidixic Acid, but also have Chloramphenicol resistance .
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       This part we use R0010 promoter ,it is to say in the absence of LacI protein and CAP protein, it promotes transcription,in the presence of LacI protein and CAP protein, it inhibits transcription. When induced by IPTG, the protein expression begin.  
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  </b><center><img src='https://static.igem.org/mediawiki/2012/2/25/Deathgene1.png'></img></center></b>
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Experimental procedure:
Experimental procedure:
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Step 1: Recovery J04500+K117000+B0015,shaking it in 37℃ for 12 to 16h at 150rpm.
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Step 2: Measure the OD600 of bacterial liquid.
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Fig1. The experimental procedure of Sweet gene’s confirmation experiment
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Step 3: Use 50ul bacterial liquid which has been diluted times to coat plates.Set IPTG(none or 1mM) control.
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Step 4: Add 5ul bacterial liquid which has been diluted  times to 5ml liquid Luria-Bertani medium. Set IPTG(none or 1mM) control. After cultivate in 37℃ shaking(150rmp) for 12 to 16h,measure the OD600 of bacterial liquid .
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Step 5: Use 50ul bacterial liquid(from step 4) which has been diluted  times to coat plates. Set IPTG(none or 1mM) control.
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Step 6: Compare bacterial liquid concentration and petri dishes on the growth of the colony to judge lethal gene’s effects .Compare the bacterial concentration and Petri dish colony growth to judge the effect of lethal gene. Judging from the OD600 of bacterial liquid and the growth state of bacteria in Petri dish to see whether the death gene work or not.
   
   
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</b><center><img src='https://static.igem.org/mediawiki/2012/0/06/Deathgene2.png'></img></center></b>
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Fig 1. The experimental procedure of Death gene’s confirmatory experiment
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Fig2. The transmission electron microscope image of conjugation
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The data of Sweet gene’s confirmatory experiment version 1.0
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  </b><center><img src='https://static.igem.org/mediawiki/2012/9/91/Deathgene3.jpg'></img></center></b>
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Fig3. The TEM image of conjugation
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Fig 2. The growth state of bacteria in Petri dish
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The data of Sweet gene’s confirmation experiment version 1.0
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(Notes: d/e/f means the bacterial liquid were diluted 104/105/106times,A/B means the concentration of IPTG none/1mM )
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</b><center><img src='https://static.igem.org/mediawiki/2012/e/ef/Deathgene4.jpg'></img></center></b>
   
   
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Fig4. The agar Luria-Bertani medium of DH5α
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Fig 3. .The comparation of colony growth
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Fig 5. The agar Luria-Bertani medium of DH5α
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Fig 6. The agar Luria-Bertani medium of BL21
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Fig7. The agar Luria-Bertani medium of BL21
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Fig8. The agar Luria-Bertani medium of HB101
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Fig9. The agar Luria-Bertani medium of HB101
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Fig10. The agar Luria-Bertani medium of conjugation
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Fig11. The agar Luria-Bertani medium of conjugation
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We process the picture by the model of our member Haoruo Jia. The results are as follows:
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Cell Number Total area Accuracy class P1 P2 P3 Level(0-1)
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DH5α 28 846 Nice 800 100 15 0.6
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DH5α 2 162 Nice 800 80 70 0.4
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BL21 439 8951 Good 800 80 8 0.32
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BL21 765 10421 Acceptable 800 80 3 0.18
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HB101 381 8700 Good 800 80 3 0.55
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HB101 2255 28262 Good 800 80 3 0.35
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Transconjugants 72 648 Good 200 80 5 0.2
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Transconjugants 130 1819 Good 1000 80 7 0.2
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Thus the efficiency of conjugation is: Tranconjugants/BL21 = 0.12347.
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(Notes: The left has IPTG of 1mM and the right has none)
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Latest revision as of 03:53, 27 September 2012

iGEM 2012 SEU_A Human Practice


Death gene

The confirmatory experiment of Death Gene

The confirmation experiment of Death Gene


The confirmatory experiment of Death gene.pdf
This part we use R0010 promoter ,it is to say in the absence of LacI protein and CAP protein, it promotes transcription,in the presence of LacI protein and CAP protein, it inhibits transcription. When induced by IPTG, the protein expression begin.
Experimental procedure: Step 1: Recovery J04500+K117000+B0015,shaking it in 37℃ for 12 to 16h at 150rpm. Step 2: Measure the OD600 of bacterial liquid. Step 3: Use 50ul bacterial liquid which has been diluted times to coat plates.Set IPTG(none or 1mM) control. Step 4: Add 5ul bacterial liquid which has been diluted times to 5ml liquid Luria-Bertani medium. Set IPTG(none or 1mM) control. After cultivate in 37℃ shaking(150rmp) for 12 to 16h,measure the OD600 of bacterial liquid . Step 5: Use 50ul bacterial liquid(from step 4) which has been diluted times to coat plates. Set IPTG(none or 1mM) control. Step 6: Compare bacterial liquid concentration and petri dishes on the growth of the colony to judge lethal gene’s effects .Compare the bacterial concentration and Petri dish colony growth to judge the effect of lethal gene. Judging from the OD600 of bacterial liquid and the growth state of bacteria in Petri dish to see whether the death gene work or not.
Fig 1. The experimental procedure of Death gene’s confirmatory experiment The data of Sweet gene’s confirmatory experiment version 1.0
Fig 2. The growth state of bacteria in Petri dish (Notes: d/e/f means the bacterial liquid were diluted 104/105/106times,A/B means the concentration of IPTG none/1mM )
Fig 3. .The comparation of colony growth (Notes: The left has IPTG of 1mM and the right has none)
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