*HIT-harbin 2012招新登记表

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             邮件标题统一为“iGEM报名”。

Before week 1

We read a lot of literature on synthetic biology and take several times of brainstorms but do not have any experiments for our summer holiday will not begin until July 18th. Several ideas have come up to our mind and we finally decide to do something about yogurt, which belongs to the track of food & energy.

Week 1 and week 2 (July 18th- July31th)

Kits, laboratory drugs and laboratory items are prepared during these days. And primers, genes which have been optimized are being synthetized by the company of Life Technologies in Shanghai. Besides, we take part in the iGEM 2011 China Meetup in University of Science and Technology of China, Hefei, Anhui, China.

Week 3 (August 1th- August 7th)

We try to extract the plasmid pMG36e from JM109. It takes us nearly one week to finish the work for the plasmid is a low copy rate one in E.coli.

Week 4 (August 8th- August14th)

PCR to test whether the plasmid is pMG36e. Result: it is the suitable large in 1.0% agarose gel electrophoresis.

Extract the plasmids with nisI gene for L.bulgaricus and the rcfB+lacR gene.

Week 5 (August 15th- August 21th)

PCR to test the plasmid with nisI gene for L.bulgaricus and the rcfB+lacR gene. Result: it is the plasmids we wanted.

Digest the plasmids with EcoRI and PstI and try to get the pars through gel extraction. Result: successfully get the parts.

Prepare competent cell of DH5α

Ligate nisI gene for L.bulgaricus with the rcfB+lacR gene. Result: failure. No colony in the petri dishes

Week 6 (August 22th- August 28th)

Try to ligate nisI gene for L.bulgaricus with the rcfB+lacR gene again. Result: successful.

Pick one colony to cultivate and then extracte the plasmid to test. Result: the liagtion is successful.

Week 7 (August 29th- September 4th)

Extract the plasmids with nisI gene for S.thermophilus and the collagen gene.

PCR to test the plasmids with nisI gene for S.thermophilus and the collagen gene.

Week 8 (September 5th- September 11th)

Ligate nisI gene for S.thermophilus and the collagen gene.

Week 9 (September 12th- September 18th)

Ligate the two recombinant parts with backbone pSB1C3. Result: failure.

Week 10 (September 19th- September 25th)

Week 11 (September 26th- October 2th)

Week 12 (October 3th - October 9th)

Week 13 (October 10th - October 16th)

The Composition Of Media

LB Medium

Yeast extact 5g

Peptone 10g

NaCl 10g

Agar 1-2%

Distilled water 1000ml pH 7.0

Boil the mixture in autoclave at 121℃ for 30min

Range of applicationb: Escherichia.coli

MRS Medium

Peptone 10g

Beef extract 10g

Yeast extact 5g

K₂HPO₄ 2g

Tween 80 1ml

Diamine citrate 2g

Na-acetate 5g

MgSO₄.7H₂O 0.5g

MnSO₄.H₂O 0.25g

Glucose 20g

Agar 1-2%

Distilled water 1000ml Adjust pH to 6.4-6.8

Boil the mixture in autoclave at 115℃ for 20min

Range of application: Lactobacillus bulgaricus

2.5% glycocoll SMRS is MRS supplemented with 2.5% glycocoll 0.5mol/L saccharose, 20mmol/L MgCl₂,20mmol/L CaCl₂

SMRSMC is MRS supplemented with 0.5mol/L saccharose, 20mmol/L MgCl₂,20mmol/L CaCl₂

Hogg-Jago glucose broth(HJG) medium

Tryptone 30g

Yeast extract 10g

Beef extract 2g

KH₂PO₄ 5g

Glucose 5g

Agar 1.5%

Distilled water 1000ml

Boil the mixture in autoclave at 115℃ for 20min

Range of application: Streptococcus thermophilus

HJGL is Hogg-Jago glucose broth supplemented with 0.5% lactose, whereas HJGLS is HJGL supplemented with 0.4M D-sorbitol

Agar plates were prepared by adding 1.5% (wt/vol) agar to the media

Competent Cell and Electroporation

The competent cell of Lactobacillus bulgaricus

• An overnight culture grown at 37°C was inoculated in 2.5% glycocoll SMRS as 2% (37°C) and incubated until the optical density at 600 nm (OD600) was 0.5. Cells were harvested by centrifugation (4,000 ×g for 10 min at 4°C) and washed twice with 1 volume of ice-cold electroporation buffer (1 mM KH₂PO₄, 0.5 M saccharose , 1 mM MgCl₂). Finally, pelleted cell were resuspended in 2ml ice-cold electroporation buffer, divided into aliquots and frozen in an ethanol-dry ice bath. Electrocompetent L.bulgaricus cells were stored at -80°C.

• Electrocompetent cells were thawed on ice, and an 50μl cell suspension was mixed with 1μg of recombinant plasmid DNA. After incubation for 30min on ice, the cells were transferred to an electroporation cuvette with a 0.2cm gap between the elec- trodes. A single pulse of 1.2kV lasting 2.5ms was delivered. The electroporated cells were immediately resuspended in 950μl SMRSMC and incubated for 2h at 37°C, before they were spread on MRS plates containing 2μg/ml erythromycin. Transformants were picked following 24 to 48h of incubation at 37°C.

The competent cell of Streptococcus thermophilus

• Preparation of electrocompetent S. thermophiluscells. An over night culture grown at 37°C was diluted 100-fold in preheated HJG (37°C) and incubated until the optical density at 660 nm (OD660) was 0.3. The culture (50ml) was then diluted 1:1 in prewarmed HJG containing 20% glycine. Afte incubation at 37°C for 1 h, cells were harvested by centrifugation (4,000 ×g for 10 min at 4°C) and washed twice with 1 volume of ice-cold electroporation buffer(5 mM KHPO4, 0.4 M D-sorbitol, 10% glycerol; pH 4.5). Finally, pelleted cell were resuspended in 4 ml ice-cold electroporation buffer, divided into aliquots and frozen in an ethanol-dry ice bath. Electrocompetent S. thermophilus cells were stored at -80°C.

• Electrocompetent cells were thawed on ice, and an 80μl cell suspension was mixed with 1μg of recombinant plasmid DNA. After incubation for 30min on ice, the cells were transferred to an electroporation cuvette with a 0.1-cm gap between the electrodes. A single pulse of 1.6kV lasting 2.5ms was delivered. The electroporated cells were immediately resuspended in 1 ml of ice-cold HJGLS and incubated for 3h at 37°C, before they were spread on HJGL plates containing 2μg/ml erythromycin. Transformants were picked following 24 to 48h of incubation at 37°C.

Note: Keep everything on ice and add all volumes in a PCR tube.

10 μL 5x buffer(Mg²⁺ plus)

4μL dNTPs

1.0μL forward primer

1.0μL reverse primer

1.0μL template (10pg-1ng)

0.5μL DNA polymerase

ddH₂O up to 50.0μL

Based on primers, set an appropriate annealing temperature

• Prepare a 1% weight-to-volume agarose gel and add SYBR dye or ethidium bromide to stain DNA.

• Place the gel in the apparatus rig with the wells facing the negative end (black-colored).

• Fill the rig with 1x TBE buffer.

• Load 2µL of 1kb ladder.

• Add 2µL of 6x loading dye to each PCR reaction tube. Load 20µL in wells.

• Run at 120V.

• Cut out the DNA fragment from the agarose gel with a razor blade, while minimizing the size of the gel slice.

• Weigh the gel slice and add 3 volumes of Buffer QG to every 1 volume of gel(100mg = 100µL).

• Dissolve the gel slice using a 60°C heat block.

• Apply the dissolved gel to the QIAquick column and centrifuge at 13,000rpm for 1 minute.

• Discard the flow-through and repeat Step 4 until all sample has passed through the column.

• Add 750µL of rinse buffer to the QIAquick column and centrifuge at 13,000rpm for 1 minute.

• Add 500µL of rinse buffer to the QIAquick column and centrifuge at 13,000rpm for 1 minute.

• Wash the column with 750µL of Buffer PE and centrifuge at 13,000rpm for 1 minute.

• Discard the flow-through and centrifuge at 13,000rpm for 2 minute to remove residual EtOH.

• Transfer the QIAquick column to a new Eppendorf.

• Add 50µL elution buffer to the center of the column and wait at least 2 minutes.

• Centrifuge at 13,000rpm for 1 minute.

The restriction enzymes we use are from Fermentas. And all the steps are according to the instruction book.