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Team:USTC/Standard & Protocol

Contents 1 Team:USTC/Standard & Protocol 1.1 Protocol 1.1.1 Standard protocol 1.1.1.1 Minipreps 1.1.1.2 Digestion 1.1.1.3 Gel Extraction 1.1.1.4 Ligation 1.1.1.5 Transformation 1.1.1.6 Colony PCR 1.1.2 Measurement protocol 1.1.2.1 Constitutive promoter measurements 1.1.2.2 Hybrid promoter response to AHL 1.1.2.3 Hybrid promoter response to AHL&aTc 1.1.2.4 M9 Medium 1.2 Standard

Protocol

Standard protocol

Minipreps

Performed with BIO BASIC INC. EZ-10 Spin Column Plasmid DNA MiniPreps Kit BS414

Digestion

The digestion enzymes we use are listed:

Pst I Fermentas ER0611 3000U

EcoR I Fermentas ER0271 5000U

Spe I Fermentas ER1251 1500U

Xba I Fermentas ER0681 400U

Gel Extraction

Performed with BIO BASIC INC. EZ-10 Spin Column DNA Gel Extraction Kit BS354

Ligation

For short segments:

TakaRa DNA Ligation Kit Ver2.0 Code D6022

BIO BASIC INC. FAST LIGATION KIT BS512

For long segments ligation:

TaKaRa DNA Ligation Kit LONG Code D6024

Transformation

Colony PCR

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Measurement protocol

Constitutive promoter measurements

1. Streak a LB plate of the strain which contain one of the parts listed in pSB1A3 .

2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic( Ampicillin 0.1mg/ml) with single colony from the plate.

3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm.

4. Cultures were diluted 1:100 into 3ml fresh medium and grown for 3hrs.

5. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance (HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell,path length 10mm,600nm,1.5 nm slit width) every 30 minutes in the next 4hrs.

Hybrid promoter response to AHL

1. Streak a LB plate of the strain which contain one of the parts listed in pSB1A3 .

2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic(Ampicillin 0.1mg/ml) with single colony from the plate.

3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm.

4. Cultures were diluted 1:1000 to tubes of 3ml fresh medium and grown for 4.5hrs.

5. Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine is diluted and added to different tubes to yield different final concentrations (1E-5,1E-7,1E-8,1E-9,1E-10M).To ensure the same response time , the AHL should be added with a time interval of 2mins between tubes, so do the measurements procedure.

6. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance ((HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell path length 10mm,600nm,1.5 nm slit width) for the first time 30 minutes after adding AHL. Repeat measurement every 30 mins in the next 4hrs.

Hybrid promoter response to AHL&aTc

1. Streak a LB plate of the strain which contain one of the parts listed in pSB1A3 .

2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic(Ampicillin 0.1mg/ml) with single colony from the plate.

3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm.

4. Cultures were diluted 1:1000 to 11 tubes 3ml fresh medium and grown for 4.5hrs.

5. Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine and aTc (anhydrotetracycline) are diluted and added to different tube to get different final concentrations listed in the table below:

Tube No.	1	2	3	4	5	6	7	8	9	10	11
c(AHL)/M	0	1.00E-06	1.00E-06	1.00E-06	1.00E-06	1.00E-06	1.00E-04	1.00E-04	1.00E-04	1.00E-04	1.00E-04
C(Atc)/ng/ml	0	0	2	20	200	2000	0	2	20	200	2000

To ensure the same response time , the AHL and aTc should be added with a time interval of 2mins between tubes, so do the measurements procedure.

6. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance ((HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell path length 10mm,600nm,1.5 nm slit width) for the first time 30 minutes after adding AHL and aTc. Repeat measurement several hours a time until OD600 reach to 0.8,it will take about 7hours in average.

M9 Medium

M9 media: for 1L 1X media

1 X M9 salt:

Na₂2HSO₄ 33.9g/L

KaH₂SO₄15g/L

NaCl 2.5g/L

NH₄Cl 5g/L

1. Dissolve 11.3 g Bacto M9 minimal salts in 970mL water.

2. Autoclave to sterilize. 121°C for 20 minutes.

8 mL 50% glycerol

1. Add 4 mL glyerol to 4 mL of H₄O

2. Filter sterilize.

2ml 1mol/L MgSO₄4

Dissolve 24.65g MgSO₄4.7H₂O in 100ml water.

Standard

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