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| - | {{Team:USTC-China/temp}}
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| - | {{Team:USTC-China/temp/top}}
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| - | {{Team:USTC-China/temp/bar4}}
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| - | 2011-5-24
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| - | ----
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| - | <div id="textNot">
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| - | by [[Yinghong Lan]][[File:110526-sm1551-pcr cheZ 1-2-2-3-3-4-4.jpg|200px|right|border]]
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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 [https://2011.igem.org//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 [https://2011.igem.org//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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| - | ----
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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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| - | ----
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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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| - | ----
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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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| - | ----
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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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| - | process PCR with plasmids aptamer-CheZ
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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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| - | ----
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| - | ligate aptamer-CheZ with PSB1C3 plasmids
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| - |
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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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| - | 7.11
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| - | transform PSB1C3 containing aptamer-CheZ into bacteria and cultivate.
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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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| - | pick up single colony from bacteria cultivated yesterday.
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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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| - | 7.30-8.3
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| - | ----
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| - | PCR of LuxPR-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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| - | </div>
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