Protocols

Liquid:

• 10 g of tryptone,
• 5 g of yeast extract,
• 10 g of NaCl,
• 1 liter of distilled water

Solid:

• 10 g of tryptone,
• 5 g of yeast extract,
• 10 g of NaCl,
• 15 g of agar,
• 1 liter of distilled water

Biobrick Assembly Restriction Digest:

For a double digest:

1. To a pcr tube, add 15 ul of miniprepped plasmid or purified PCR product
2. Add 3 ul of the appropriate buffer
3. Add 1ul of each enzyme.
4. Incubate at 4˚C overnight.

Biobrick Assembly Ligation:

1. In a PCR tube, add restriction digested and PCR purified insert to backbone in a 4:1 molar ratio, usually 1 ul backbone with 4 ul insert works.
2. Add 10ul T4 ligase buffer
3. Add 1 ul T4 ligase
4. Create a negative control replacing the insert with water
5. Add reactions to thermal cycler, and leave at 4˚C overnight.
6. Use 2 ul of the ligation reactions to transform cells.

PCR/Gel Extraction

Fresh TAE buffer as running buffer is recommended. Reusing running buffer will result the increase of the pH and then reduce yields.

1. For cycle-pure (PCR reaction): Add 2 volumes of Buffer GC to 1 volume of the PCR reaction and mix completely by vortexing. Briefly spin the tube to collect any drops from the inside wall and tube lid. For PCR products less than 200 bp, add 5 volumes of Buffer GC to 1 volume of PCR reaction
2. For agarose gel: Excise the DNA fragment from the agarose gel and weigh it in a 1.5 mL microtube. Add 1 volume of Buffer GC to 1 volume of gel to the 1.5 mL microtube and incubate the mixture at 55-60°C for 8 min. Mix the tube by tapping the bottom every 2 min till the gel has melted completely. Cool the tube to room temperature. Note: A gel slice of 100 mg approximately equals to 100 μL. For DNA fragment less than 200 bp, add 1 volume of isopropanol.
3. Transfer up to 700 μL DNA/Buffer GC mixture to a spin column with a collection tube. Centrifuge at 13,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube. Repeat this step to process the remaining solution.
4. Add 300 μL of Buffer GC into the DNA Mini Column. Centrifuge at 13,000 x g for 30 to 60 seconds at room temperature to wash the DNA Mini Column.
5. Add 650 μL DNA Wash Buffer to the column and centrifuge at 13,000 x g for 30 s at room temperature. Discard the flow through and insert the column, with the lid open, back to the collection tube. Repeat step “4”. Note: Ensure that ethanol has been added to DNA Wash Buffer as instructed .
6. Centrifuge the empty DNA column, with the lid open, at 13,000 x g for 2 min to dry the ethanol residue in the matrix. Note: The residual ethanol will be removed more efficiently with the column lid open during centrifugation.
7. Place the column into a clean 1.5 mL micocentrifuge tube and add 30-50 μL pre-warmed (60oC) Elution Buffer or ddH2O to the center of the column. Incubate at room temperature for 1 min. Centrifuge at 13,000 x g for 1 min

Miniprep Plasmids

1. Inoculate 1-4 mL LB containing appropriate antibiotic with a fresh colony from a freshly streaked selective plate. Incubate at 37°C for 14-16 hours with vigorous shaking. Note: Prolonged incubation (> 16 hours) is not recommended since the E.coli starts to lyse and the plasmid yields may be reduced. Do not grow the culture directly from the glycerol stock. This protocol is optimized for E. coli strain cultured in LB medium. When using TB or 2xYT medium, special care needs to be taken to ensure the cell density doesn’t exceed 3.0 (OD600). Buffers need to be scaled up proportionally if over amount of cultures are being processed.
2. Harvest the bacterial culture by centrifugation for 1 min at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely. Note: Residue medium will cause,
   • Poor cell lysis and thus lower DNA yield.
   • Loose pellet after centrifugation in step 6.
3. Add 250 μL Buffer A1 (Add RNase A to Buffer A1 before use) and completely resuspend bacterial pellet by vortexing or pipetting. Note: Complete resuspension is critical for bacterial lysis and lysate neutralization.
4. Add 250 μL Buffer B1, mix gently by inverting the tube 10 times (do not vortex), and incubate at room temperature for 5 minutes. Note: Do not incubate for more than 5 minutes. Note: Buffer B1 precipitates (cloudy look) below room temperature. Warm up Buffer B1 at 50°C to dissolve precipitation before use.
5. Add 350 μL Buffer N1, mix completely by inverting/shaking the vial for 5 times and sharp hand shaking for 2 times. Note: Incubating the lysate in ice for 1 min will improve the yield. It is critical to mix the solution well. If the mixture still appears conglobated, brownish or viscous, more mixing is required to completely neutralize the solution.
6. Centrifuge the lysate at 13,000 rpm for 10 minutes at room temperature. Note: If the lysate doesn’t appear clean, reverse the tube angle, centrifuge for 5 more minutes and then transfer the clear lysate to DNA column.
7. Carefully transfer the clear lysate into a DNA column with a collection tube, avoid the precipitations, spin at 13,000 rpm for 1 minute, discard the flow-through and put the column back to the collection tube.
8. Optional: Add 500 μL Buffer KB into the spin column, centrifuge at 13,000 rpm for 1 minute. Remove the spin column from the tube and discard the flow-through. Put the column back to the collection tube. Note: Buffer KB is recommended for endA+ strains such as HB101, JM101, TG1 or their derived strains. It is not necessary for isolating DNA from endA- strains such as Top 10 and DH5a.
9. Add 650 μL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 13,000 rpm for 1 minute at room temperature. Remove the spin column from the tube and discard the flow-through. Repeat step “9” to improve the recovery.
10. Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 2 minutes at 13,000 rpm. Note: Residual ethanol can be removed more efficiently with the column lid open. It is critical to remove residual ethanol completely.
11. Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 μL (> 50 μL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again. Note: If ddH2O is applied, please make sure the pH is no less than 7.0 (7.0-8.5 is preferred). NaOH could be used to adjust the pH of ddH2O. The DNA is ready for downstream applications such ascloning/subcloning, RFLP, library screening, in vitro translation, sequencing, transfection of robust cells such as HEK293 cells. Note: It’s highly recommended to remove the endotoxin (PD1212) if the DNA is used for endotoxin-sensitive cell lines, primary cultured cells or micro-injection.
12. The DNA concentration can be calculated as follows, Concentration (μg/mL) = OD260nm x 50 x dilution factor.

Transforming DNA from Distribution Plates:

1. Thaw competent cells on ice.
2. Add 10 microliters of pure water to each well of DNA from plates, pipette up and down.
3. Transfer into storage tube.
4. Pipette 1-2 microliters of the DNA into the competent cell tubes.
5. Stir with pipette tip, gently flick tube.
6. Leave on ice for 30 minutes.
7. Heat Shock for 45 sec by using a water bath set to 42°C and then chill on ice for 2 min.
8. Pipette 500 micro Liters of S.O.C. (LB + glucose) into 14ml culture tubes; transfer the competent cells into these tubes and incubate in a 37 degree shaker for 40-60 minutes before plating.
9. For source plate DNA, plate 100 microliters.

Taq PCR

Set up tubes with 7 ul H20, 1 ul template, 1 ul forward primer, 1 ul reverse primer, 10 ul PCR mix

• 94°C for 1:30 min
• 35 cycles of:
  • 94°C for 0:30 min
  • 54°C for 1:00 min
  • 72°C for 1:00 min/Kb
• Final extension: 72°C for 6:00 min

Colony PCR

1. Suspend colonies in 5 ul H20
2. Lyse with 98°C incubation for 10 minutes
3. Use 1 ul of this suspension as template
4. Set up tubes with 7 ul H20, 1 ul template, 1 ul forward primer, 1 ul reverse primer, 10 ul PCR mix

Cycle:

• 2 minutes at 98°C
• Run 30 cycles of:
  • 10 seconds at 98°C
  • 30 seconds at 55°C-65°C
  • 40 seconds at 72°C
• Final extension: 5 minutes at 72°C

eFusion Cloning Kit

Cloning Materials Preparation

• Vector Construction: Use any one or two restriction enzyme to linearly cut the vector.
• Target Fragment Construction: Add nucleotide sequences same as ones from the sticky ends to the 5' terminus of every PCR primer.

Recombination of Goal Segments and Vector

Mix inserted segments, vector and sFusase for 30min at room temperature. Then put the mixture on the ice and prepare to transform.

Mixture:

• H20 X mL
• PCR products 100-10ng X mL
• Vector 100-200ng X mL
• eFusase 3mL
  • Total 15mL

Tranformation.

March

2012-3-18 Sun

After discussion in the seminar, 13 students organized the iGEM team and set out to design our project.

2012-3-24 Sat

Our project application were admitted in our school and the registry work started.

The achievements of one week's work:

Muyuan Chen & Xuelong Fan: responsible for the fund application and team registry work.

Xingyu Zhou: designed the bio-sensor based on the asymmetric colony.

Ying Cheng & Zhu Zhu: Searched the information about quorum sensing.

Xiaodan Xu: Read the literature on E.coli's flagella and division.

Peng Cheng & Le Zhou: designed the wiki and logo.

2012-3-25 Sun

Shen Gao: improved the method of cultivation of E.coli.

2012-3-26 Tue

Muyuan Chen: Did some research on light sensor. We voted and named our team SEU_Omega~

2012-3-27 Wed

Filled the application form on the www.igem.org.

2012-3-31 Sat

Xintong Ling: Finished the simulation of diffusion equation in the rectangular case.

April

2012-4-1 Sun

Xintong Ling: finished the diffusion model based on the random containers and made a plan for further models.

2012-4-5 Thur

Muyuan Chen: Made the schedule and experiment program before our summer vacation. Found some information about toggle switch.

Xuelong Fan: Did some research on the light sensor.

Xiaodan Xu: Learnt about how to use NCBI.

2012-4-7 Sat

Had a group meeting and discussed about the future plans.

Xiaodan Xu: Found out the antisense FtsZ gene can be used to control the shape of E.coli's colony.

2012-4-9 Mon

Shen Gao: Choose Pet-32a plasmid for our experiment.

2012-4-12 Thur

Xiaodan Xu: Found a way to verification the primers.

2012-4-13 Fri

Xintong Ling: Finished the simple cell division model.

2012-4-17 Tue

Shen Gao: Learnt about the primer design.

Xingyu Zhou: Designed the quorum sensing part——the AHL system.

Muyuan Chen: Use vectorNTI to analysis plasmid.

2012-4-19 Thur

Xintong Ling:finished the Cellular Automata based on the AHL density.

Xingyu Zhou:Made the expriment plan for the AHL system and found some related kits for his plan.

2012-4-20 Fri

Muyuan Chen: Made the experiment plan for the antisense FtsZ.

Xintong Ling:improved the model of cellular automata.

2012-4-22 Sun

Shen Gao:improved the method of gel extraction and agarose gel electrophoresis.

Xingyu Zhou:Made a summary of AHL sensing system.

2012-4-24 Tue

Xintong Ling & Kai Gui:made the schedule for the further modeling.

Kai Gui:finished the model of biosensor.

2012-4-26 Thur

Xiaodan Xu:Made the experiment plan for antisense FtsZ.

2012-4-29 Sun

Xiaodan Xu:Made a list of the protection base for the restriction sites.

May

2012-5-2 Wed

Xintong Ling: modified the model of biosensor.

2012-5-3 Thur

Xiaodan Xu:Chose the target fragment of FtsZ.

2012-5-7 Mon

Muyuan Chen & Xiaodan Xu: Modified the experiment program--the promoter and the hairpin of the antisense fragment.

2012-5-8 Tue

Ying Cheng: Made the summary of the previous project.

Kai Gui: Finished the model of cellular automata based on the AHL signal diffusion.

2012-5-10 Thur

Our group discussed about the human practice.

Xingyu Zhou:Modified the experiment program of AHL part.

Xintong Ling: Summarise the previous model.

Xiaodan Xu: sorted out the kits list.

2012-5-11 Fri

Xintong Ling:modified the model of cellular automata.

2012-5-12 Sat

Xingyu Zhou:finished the complete AHL experiment program.

2012-5-13 Sun

Xiaodan Xu: Made a list of possibly useful kits.

2012-5-14 Mon

Muyuan Chen: Ordered the paired termini sequence.

2012-5-16 Wed

Xintong Ling & Kai Gui & Jun Zhang: had a meeting talking about the model.

2012-5-22 Tue

Xingyu Zhou: Found out the chemical way of the AHL detection.

2012-5-24 Thur

Xiaodan Xu: transformed the K082034 to make sure the method of Kit usage is correct.

2012-5-27 Sun

Had a group meeting and summarise the work we had done.Made the schedule for the summer vacation and everyone started the preparation for their final examinations.

2012-5-31 Thur Xingyu Zhou: Finished the budget for our project.

June Week 3

2012-6-12 Tue

We got the paired termini fragment in the PUC vector.

2012-6-16 Sat

Muyuan Chen:wrote the experiment program of AHL.(BBa_T9002+F1610)

June Week 4

PCR: FtsZ target fragment

Transform: F1610 R0010

Recovery: I13521

2012-6-19 Tue

Check: F1610 failed; R0010 growed

Digestion: FtsZ1 FtsZ5 paired termini

Transform: F1610

Culture: R0010

Results: AHL failed

2012-6-20 Wed

Check: F1610 failed again

Transform FtsZ1/5+paired termini

Results: AHL failed

2012-6-21 Thur

Check: None

Miniprep plasmids R0010

Transform FtsZ1/5+paired termini

Culture: F1610; C0261; B0015

Others Found our instructor Zuhong Lu and reported our schedule.

2012-6-22 Fri

Check: F1610 FtsZ5+paired termini failed

PCR: Paired termini

Transform: J5526; I13202; T9002

Culture: FtsZ1+paired termini; T9002

Results: AHL failed; We got ftsZ1+termini~

2012-6-23 Sat

Check: J5526 I13202 growed.

Miniprep plasmids T9002

Culture: J5526; I13202

Others Muyuan Chen : Designed the toggle switch. BBa_K415301 BBa_K415300

June Week 5

Miniprep plasmids: I13202; J5526

Transform: I13202; K415023; K091230

Recovery: Pet32a

Culture: I13202+T9002

2012-6-26 Tue

Check: K415023 failed

Digestion: T9002; I13202

Transform: K415023

Recovery: I13202

Culture: I13202; K091230

Results: Toggle switch failed

2012-6-27 Wed

Digestion: T9002+I13202

Transform: R0082; K415301; M30109; K415023; K091230

Recovery: T9002

2012-6-28 Thur

Check: T9002 failed

Ligation: T9002+I13202

Recovery: J5526

Culture: R0082 K415301 M30109 K415023 K091230

Results: AHL maybe failed.

2012-6-29 Fri

Check: R0082 K415023 failed

Miniprep plasmids: K415301; M30109; K091230

Digestion: Pet32a; paired termini

Ligation: Pet32a+paired termini

Transform: R0082; R0083; R0084

Results: AHL maybe would failed.

2012-6-30 Sat

Check: R0082 R0083 R0084 failed

Miniprep plasmids: Pet32a

Digestion: ftsZ +paired termini; Pet32a

Ligation: ftsZ+paired termini+pet32a

Recovery: Pet32a+egfp

Culture: FtsZ1 +Paired termini

Results: Light sensor failed

July Week 1

2012-7-1 Sun

Check: The recovery was fine

Ligation: FtsZ+Paired termini+pet

Transform: FtsZ+Paired termini+pet

Others: Xingyu Zhou: found the way of AHL detection.

Xiaodan Xu: Supplement the knowledge on the toggle switch.

2012-7-2 Mon

Check: We got FtsZ1+pt+pet (not confirmed)

PCR: R0082;

• The marker failed. We are not sure about the result.

Ligation: FtsZ+Paired termini+pet

Transform: Ftsz1+pt+pet R0082 R0083 I13507 J5526 J4450 R0011

Recovery: T9002 I13202 M30109

Culture: R0084 ftsz+pt

2012-7-3 Tue

Check: The FtsZ5 failed. J5526 didn’t turn red.

Miniprep plasmids: T9002 I13202 M30109 R0084 FtsZ1

• ftsZ1 R0084 failed

PCR: T9002;

• The density was too low.

Digestion: T9002+I13202

Transform: p0451 r0051 i732820 i13973

Recovery: R0083 I13507 R0011 R0084 FtsZ1 J04450

Others Muyuan Chen: Sorted out all the kits we might need.

2012-7-4 Wed

Check: I13507 failed

Miniprep plasmids: R0011 R0084 FtsZ1 J04450 r0083

• Only J04450 R0084 FtsZ1 succeeded.

PCR: P0451 I732830 R0051 I13973 R0084

• Didn’t get R0051

Ligation: FtsZ+Paired termini+pet

Transform: I13507 J23039 R0062 J37033

Culture: P0451 i732820 R0051 I13973

2012-7-5 :Thur

Check: R0051 failed

Miniprep plasmids: P0451 I732820 I13973 R0084 M30109 I13202 T9002

• R0084 M30109 failed

PCR: I13507 R0062 J37033 J23039 T9002

Digestion: T9002 I13202 R0084 M30109

Transform: R0051 R0011

Recovery: Paired termini Pet

Others Xintong Ling: Started the simulation of colony.

2012-7-6 Fri

Check: The transformation failed all! We didn’t find out the cause.

Miniprep plasmids: I13973 J37033 J23039 R0084 M30109 I13507 Paired termini pet32a

• R0084 M30109 failed

PCR FtsZ1/5 r0062

• All succeeded

Digestion: Pet+paired termini

Ligation: T9002+I13202

Transform: R0051 R0011

Others:

• We had a short meeting, talked about our project again.
• Today the lab had power failure at noon.

2012-7-7 Sat

Check: none failed

Miniprep plasmids: R0084 R0062

• We got the two plasmids

PCR: R0051 R0011

• R0011 failed

Ligation: PET+Paired termini

Transform: T9002+I13202

Others Xingyu Zhou: Sorted out the experiment protocols.

Kai Gui: figured out the diffusion parameters.

2012-7-8 Sun

Check: T9002+I13202 failed.

Our holiday~