Method

iGEM UT-Tokyo

Dual luciferase assay

Preparation

Dual-GloR Luciferase Assay System (Promega)

1. Transfer the contents of one bottle of Dual-GloR Luciferase Buffer to one bottle of Dual-GloR Luciferase Substrate to make Dual-GloR Luciferase Reagent.
2. Calculate the amount of Dual-GloR Stop & GloR Reagent needed for the experiment. Using a new container, dilute the Dual-GloR Stop & GloR Substrate 1 : 100 into Dual-GloR Stop & GloR Buffer to make the needed volume of Dual-GloR Stop & GloR Reagent.

PBS

Luminescence vial

Protocol

1. Centrifuge the incubative tube at 3,000g for 15 min with soft brake.
2. Decant supernatant, wash with 1mL PBS
3. Centrifuge at 3,000g for 5 min with soft brake.
4. Decant supernatant, add 100 ul cell lysis buffer.
5. Remove lysate 10-50 ul to the vial.
6. Add a volume of Dual-GloR Reagent equal to the volume of cell lysate.
7. Wait at least 5 minutes to allow for chemiluminescence to become stable, then measure the firefly luminescence in a uminometer.
8. Add a volume of Dual-GloR Stop & GloR Reagent equal to the original culture medium volume.
9. Wait at least 5 minutes, then measure Renilla luminescence
10. Calculate the ratio of luminescence from the experimental reporter to luminescence from the control reporter.

Notes

Cell diffsion assay

Preparation

0.25% agar LB plate (0, 1, 10, 40 or 100uM IPTG)

Protocol

1. Pick single colony(cheZ-, cheZ^res or cheZ^rep) using a pipetman with sterile tip.

   cheZ^res is the cheZ- transformed with lacP-RBS-CheZ-d.Ter.

   cheZ^rep is the cheZ- transformed with cIP-RBS-CheZ-d.Ter-lacP-RBS-cI Represser-d.Ter.

2. Inoculate tip with colony into 0.25% agar plates (0, 1, 10, 40 or 100uM IPTG).
3. Incubate at room temperature (25°C).
4. Exposure the plates every 24 hours.

Notes

0.25% agar plate is fragile. You should keep it horizontal.

L-aspartate chemotaxis assay

Preparation

0.25% agar LB plate

10mM L-Asp solution

Protocol

1. Pick JM109 single colony using a pipetman with sterile tip.
2. Inoculate tip with the colony into the point that is 25mm distant from the center of 0.25% agar LB plate.
3. Incubate at room temperature (25°C) for 45 hours.
4. Trickle 40uL of 10mM L-Asp solution in its center(Asp+) or 40uL of MilliQ(Asp-).
5. Exposure the plates after inoculating at room temperature for more 20 hours.

Notes

0.25% agar plate is fragile. You should keep it horizontal.

SOS response preparation

Preparation

an UV irradiator (15W / wavelength: 254nm)

LB broth

35mm dish

Protocol

1. Culture until OD₆₀₀ is 0.4-0.6
2. Spit into dishes so that a medium depth is 4mm (about 1.4mL in a 35mm dish)
3. UV irradiation (15W / 254nm) for 30-60 sec agitating gently.
4. Transfer into tubes (1.25mL/tube) and add 2.5mL LB broth.
5. Culture for 30mm at 37 °C.

Notes

Make sure that cells do not lack RecA.

Assembly parts

Digest

Preparation

Plasmid

10x buffer (H or M)

0.1% BSA

Emzyme (EcoR I, Xba I, Spe I, Pst I)

Protocol

1. Add plasmid (≦ 2000ng total)

   MilliQ up to 30uL

   3uL 10x H or M buffer

   3uL 0.1% BSA

   0.5uL emzyme I

   0.5uL emzyme II

2. Incubation at 37 °C for more than one hour.

Notes

H buffer for E, P, ES, SP and EP cut.

M buffer for X, S, EX and XP cut.

Ligation

Preparation

Vector DNA

Insert DNA

2x Ligation Mix (from Ligation Convenience Kit by Nippon Gene)

Protocol

1. Make reaction liquid

   MilliQ up to 20uL

   10uL 2x Ligation Mix

   Vector DNA

   Insert DNA

2. Incubation at 16 °C for 15-30 min.

Notes

Vector DNA : Insert DNA (molar ratio) = 1 : 1-1.5

DNA is about 25ng total

Transformation

Making Competent E. coli cell

Preparation

LB broth

SOB broth

Mg sol.

TB

DMSO

Protocol

1. Shaking culture in 2mL LB broth from frozen stock (37 °C O.N.)
2. Add 1.0mL O.N. cuture to 80mL SOB broth(+0.8mL Mg sol.)
3. Culture until OD₆₀₀ is 0.3-0.5 (37 &deg for about 2 hours or 20 &deg for about 6 hours).
4. On ice for 10 min.
5. Split into two of 50mL tubes and centrifuge for 5 min (4 °C 4000G).
6. Remove supernatant.
7. Suspend in TB(10 mL / tube).
8. On ice for 15 min.
9. Add 200mL DMSO /tube and mix on ice.
10. On ice for10-15 min.
11. Bring together in a tube and mix well.
12. Split into tubes (100uL/tube) and quick freezing in liquid nitrogen.
13. strage at -80 °C

Notes

Transformation of E. coli

Preparation

iGEM parts / ligation products

SOC or LB (No antibiotic)　500μL

TE　15μL

plates

ice box

heat block(42°C)

competent cells

→ always on ice! Melt on ice! Mix DNA as soon as cells melt!

Protocol

to thaw out igem parts

1. With a pipette tip, punch a hole in the foil
2. Add 15μL of TE (MilliQ),and pipetting
3. Pipette 1μL of the resuspended DNA Transformation into your desired competent cells
4. Hold on ice for 30 min.
5. Heat shock at 42°C for 45 seconds (and on ice after it)
6. Add 300uL of LBborth in each epp
7. Wait for 10 mins
8. Hold at 37°C for 30 min.

   (this step can be skipped with ampicillin selection)

9. Plate out
10. Incubate at 37°C

Purification of DNA

Miniprep

Preparation

kit of Promega (SVMinipreps)

incubative tube

1.5mL epp tube

MilliQ

Procedure

1. pour contents out of the incubative tube into the 1.5mL tube as you can
2. centrifuge for 10min (15,000rpm)

   (you can centrifuge incubative tube directly when it can endure up to 6,000g )

3. throw supernatant fluid away not to damage the precipitation

   ( you should decant by using yellow tip first / remove culture medium as you can / throw waste water away in bio hazard!)

4. add 250μl cell resuspension solution (red label)、suspend completely

   (incomplete suspending decreases yields / you should use epp stand like a washboard)

5. add 250μl Cell lysis solution（green label）
6. turn the tube upside down four times slowly not to bubble
7. add 10μl Alkalin　Protease　Sol. （small bottle）
8. turn the tube upside down four times slowly not to bubble
9. wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）
10. add 350μl Neutralization Sol. (blue label）
11. turn the tube upside down four times slowly not to bubble
12. centrifuge for 10min (15,000rpm)
13. put the supernatant fluid to column (germ’s wreckage is adhering below)
14. centrifuge for 1min (15,000rpm)
15. throw flow through (the liquid in the tube below) away
16. add 750μl Wash Sol. to column and centrifuge for 1min (15,000rpm)
17. throw flow through away, put 250μL Wash Sol. to column and centrifuge for 1min (15,000rpm)
18. change the column into 1.5ml tube and centrifuge for 2min (15,000rpm)
19. change the tube into new one and add 50μL MilliQ

    (use Nucleas-Free Water in the kit instead of MilliQ)

20. centrifuge for 1min (15,000rpm) after waiting for 1min
21. take 1 to 1.5μL and determine the concentration by NanoDrop (Don’t dilute)
22. label them

Gel extraction, PCR clean-up

Preparation

Kit of promega

Gel

Procedure

1. Gel Slice and PCR Product Preparation

   Dissolving the Gel Slice

   1. Cut out gel with wanted band and put it in a tube.
   2. Add 3 parts Mem. binding sol. to 1 part Gel volume.

   Processing PCR Amplifications

   1. Add an equal volume of Membrane Binding Solution to the PCR amplification.
2. Shake & Incubate at 50-65 degrees C until gel is completly dissolved.
3. Put in the column.
4. Centrifuge at 15,000 rpm for 1 minute
5. Empty collection tube and add 700 ul Mem. Wash sol, centrifuge for 1 minute.
6. Empty collection tube and add 500 ul Mem. Wash sol, centrifuge for 1 minute.
7. Change the column to a new 1.5 ml Eppendorf and centrifuge at 15,000 rpm for 1 minute.
8. Change the old tube to a new 1.5 ml Eppendorf, add 30ul Nuclease-Free Water, incubate at RT for 1 minutes, then centrifuge at 15,000 rpm for 1 minute.
9. Measure concentration, label the Eppendorf.

Notes

Percent Recovery Versus Double-Stranded DNA Fragment Size

DNA Fragment Size	Percent Recovery
55bp	26%
100bp	84%
1,000bp	92%
23,130bp	47%

Ethanol precipitation for Parts shipping

Preparation

100%, 70% Ethanol

TE buffer

Miniprep products

Procedure

1. Add 2 volumes ice cold absolute ethanol to sample.
2. Incubate 1 hr at -80°C.

(The long incubation time is critical for small fragments)

1. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
2. Remove supernatant.
3. Wash with 750-1000 ul room-temperature 95% ethanol.
4. Centrifuge for 10 minutes at 4°C at maximum speed (generally >10000 g at least).
5. Dry the pellet. For this you can air dry (tubes open, ~15 min).

   (Overdrying can make DNA hard to re-dissolve)

6. Add 10 ul TE buffer. Vortex and spin down to resuspend.
7. Determine the concentration by NanoDrop.

Notes

Pellets can be very small and hard to see. Make sure that you don't break invisible pellets on all steps.

Reagents

Strains

JM109 (Takara Bio INC.)

• Genotype:

recA1, endA1, gyrA96, thi-1, hsdR17(rK-mk+), e14?(mcrA?), supE44, relA1, Δ(lac-proAB)/F' [traD36, proAB+, lac Iq, lacZΔM15]

BL21(DE3)

• Genotype:

F- ompT hsdSB (rB-mB-) gal dcm (DE3)

ccdB survival (invitrogen)

• Genotype:

F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG fhuA::IS2

JW 1970 (cheZ-)

derived from K12 BW25113

• Genotype

(araD-araB)567, lacZ4787(::rrnB-3), lambda-, rph-1, (rhaD-rhaB)568, hsdR514

details available at http://ecoli.aist-nara.ac.jp/GB6/info.jsp?id=JW1870

DH5α (invitrogen)

• Genotype:

F- φ80lacZΔM15 Δ(?lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 λ-thi-1 gyrA96 relA1 tonA

CJ236 (Takara Bio INC.)

• Genotype:

dut1, ung1, thi-1, relA1/pCJ105（F' camr）