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Genome Extraction of Top10

• Aim: to get the genome of Top10 for cheZ
• Time:2011-5-23
• Members: Duo An, Mengping Sun & Yinghong Lan
• Result:from the electrophoresis result, we can say we have basically got the whole genome of Top10.
• Notes: EP1:60ng/ul EP2:200ng/ul

2011.6.28 --- cultivate the bacteria of CheZ deficiency(RP1616),and the Control group(RP437). check the resistibility of RP1616 and RP437 result:both of the two groups are of none resistibility. members:Siwei Luo, Fangzhou Zhao, Zhilin Chen...

2011.6.29

check the motility of RP1616 strain and RP437 strain cultivate Top10 strain result: the bacterial plaque of RP1616 is smaller than that of RP437, implying that RP1616 is of CheZ deficiency, causing the decreased motility of the bacteria.

6.30

prepare for the competent cell of Top10 strain cultivated on 6.29 extract the genome of Top10 strain and use it as complates to run PCR of CheZ result: the concentration of PCR result is too low to continue the experiments.

7.1

conduct PCR of CheZ again ligate the CheZ DNA segment to plasmid PSb1C3, and transform it into Top10 competent cells result:如图

7.2

pick up single colony from the Petri dish to reproduce, in which the competent cells are cultivated and use them colony PCR result:The bacteria grow up well on dishes with chloromycetin resistency，verifying the transformation is successfull while the concentration of PCR product is pretty high,demonstrating that the plasmids indeed contains the sagment CheZ.

7.3

extract the plasmids from the reproduced bacteria and send it to check the base sequence result: the concentration of the plasmids extracted is about 500ng/uL

7.4

extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2010 and put them into Top10 strain by transformation (LuxPr: Plate 2,24C Terminator : Plate 1,6O) result: the bacteria on the plate with Amp resistency grows well

7.5

pick up single colony from the Petri dish to reproduce in the liquid culture medium

7.6

extract plasmids containing LuxPR and Terminator and make enzyme digestion with EcoR1 and Spe1 in order to validate the existence of the two parts recieve part Toggle Switch and rbs-Ci-ter from PKU and conduct transformation

result: Due to the short length of LuxPR and Terminator, the result of the enzyme digestion is hard to examine by electrophoresis.

7.7

result: there are a few colonies with part Toggle switch grown while no colony of rbs-Ci-ter appeared.

7.8

pick up single colony with part Toggle switch . extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2011 and put them into Top10 strain by transformation

7.9

pick up single colony with LuxPR & terminator. extract plasmids containing toggle switch to conduct PCR. result:as the picture shows. the base sequence of CheZ extracted before is proved right.

7.10

extract plasmids containing LuxPR and terminator result:the concentractions of plasmids LuxPR and terminator are both 600ng/ul

7.12

carry out Double digestion of toggle switch and pSB1c3 and ligate them together.

result: Both the location and the brightness of the electrophoretic bands correspond with our expectation.

7.14

transform the part rbs-ci-ter sent from PKU again into bacteria. check the colors of the colonies containing toggle switch. result: the ratio of red vs green is 1:1, verifying the function of toggle switch.