Team:USTC-China/Notebook

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2011-5-24
 
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<div id="textNot">
 
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by [[Yinghong Lan]]<pic>[[File:110526-sm1551-pcr cheZ 1-2-2-3-3-4-4.jpg|200px|right|border]]</pic>
 
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'''Genome Extraction of Top10'''
 
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*Aim: to get the genome of Top10 for [[https://2011.igem.org/Team:USTC-China/cheZ|cheZ]]
 
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*Time:2011-5-23
 
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*Members: Duo An, Mengping Sun & Yinghong Lan
 
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*Result:from the electrophoresis result, we can say we have basically got the whole genome of Top10.
 
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*Notes: EP1:60ng/ul EP2:200ng/ul
 
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2011.6.28
 
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cultivate the bacteria of [[/Team:USTC-China/cheZ|cheZ]] deficiency(RP1616),and the Control group(RP437).
 
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check the resistibility of RP1616 and RP437
 
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result:both of the two groups are of none resistibility.
 
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members:Siwei Luo, Fangzhou Zhao, Zhilin Chen...
 
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2011.6.29
 
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check the motility of RP1616 strain and RP437 strain
 
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cultivate Top10 strain
 
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result: the bacterial plaque of RP1616 is smaller than that of RP437, implying that RP1616 is of ''[[/Team:USTC-China/cheZ|cheZ]]'' deficiency, causing the decreased motility of the bacteria.
 
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6.30
 
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prepare for the competent cell of Top10 strain cultivated on 6.29
 
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extract the genome of Top10 strain and use it as complates to run PCR of CheZ
 
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result: the concentration of PCR result is too low to continue the experiments.
 
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7.1
 
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conduct PCR of CheZ again
 
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ligate the CheZ DNA segment to plasmid PSb1C3, and transform it into Top10 competent cells
 
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result:as the picture shows.
 
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7.2
 
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pick up single colony from the Petri dish to reproduce, in which the competent cells are cultivated and use them colony PCR
 
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result:The bacteria grow up well on dishes with chloromycetin resistency,verifying that the transformation is successfull
 
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while the concentration of PCR product is pretty high,demonstrating that the plasmids indeed contains the sagment CheZ.
 
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7.3
 
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extract the plasmids from the reproduced bacteria and send it to check the base sequence
 
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result: the concentration of the plasmids extracted is about 500ng/uL
 
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7.4
 
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extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2010 and put them into Top10 strain by transformation
 
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(LuxPr: Plate 2,24C  Terminator : Plate 1,6O)
 
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result: the bacteria on the plate with Amp resistency grows well
 
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7.5
 
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pick up single colony from the Petri dish to reproduce in the liquid culture medium
 
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7.6
 
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extract plasmids containing LuxPR and Terminator and make enzyme digestion with EcoR1 and Spe1 in order to validate the existence of the two parts
 
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recieve part Toggle Switch and rbs-Ci-ter from PKU and conduct transformation
 
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result: Due to the short length of LuxPR and Terminator, the result of the enzyme digestion is hard to examine by electrophoresis.
 
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7.7
 
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result: there are a few colonies with part Toggle switch grown while no colony of rbs-Ci-ter appeared.
 
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7.8
 
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pick up single colony with part Toggle switch .
 
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extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2011 and put them into Top10 strain by transformation
 
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7.9
 
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pick up single colony with LuxPR & terminator.
 
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extract plasmids containing toggle switch to conduct PCR.
 
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result:as the picture shows.
 
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the base sequence of CheZ extracted before is proved right.
 
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7.10
 
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extract plasmids containing LuxPR and terminator
 
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result:the concentractions of plasmids LuxPR and terminator are both 600ng/ul
 
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7.12
 
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carry out Double digestion of toggle switch and pSB1c3 and ligate them together.
 
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result: Both the location and the brightness of the electrophoretic bands correspond with our expectation.
 
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7.14
 
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transform the part rbs-ci-ter sent from PKU again into bacteria.
 
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check the colors of the colonies containing toggle switch.
 
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result: the ratio of red vs green is 8:25, verifying the function of toggle switch.
 
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7.15
 
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pick up the single colony from the plate yesterday and ten hours later extract the plasmids containing Ci-terminator.
 
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7.16
 
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conduct PCR with the plasmids yesterday to reproduce rbs-ci-Term.
 
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result:failure
 
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7.18-7.21
 
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ligate the standard part Terminator to the end of toggle switch
 
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result: we conduct a enzyme digestion with EcoR1 and Pst1 to the final ligation product and the electrophoresis result shows that the ligation is successful.
 
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7.22-7.29
 
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perform the following experiments: PCR of rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction.
 
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result: the concentration of plasmids with ligation gene is as high as ...
 
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7.30
 
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</div>
 

Latest revision as of 18:06, 5 October 2011