Team:USTC/Standard & Protocol

From 2009.igem.org

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 +
==Protocol==
 +
===Assembly 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====
 +
 +
 +
 +
----
 +
 +
===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:
 +
{| {{table}}
 +
{|align="center"
 +
| align="center" style="background:#f0f0f0;"|'''Tube No.'''
 +
| align="center" style="background:#f0f0f0;"|'''1'''
 +
| align="center" style="background:#f0f0f0;"|'''2'''
 +
| align="center" style="background:#f0f0f0;"|'''3'''
 +
| align="center" style="background:#f0f0f0;"|'''4'''
 +
| align="center" style="background:#f0f0f0;"|'''5'''
 +
| align="center" style="background:#f0f0f0;"|'''6'''
 +
| align="center" style="background:#f0f0f0;"|'''7'''
 +
| align="center" style="background:#f0f0f0;"|'''8'''
 +
| align="center" style="background:#f0f0f0;"|'''9'''
 +
| align="center" style="background:#f0f0f0;"|'''10'''
 +
| align="center" style="background:#f0f0f0;"|'''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:
 +
 +
Na<sub>2</sub>2HSO<sub>4</sub> 33.9g/L
 +
 +
KaH<sub>2</sub>SO<sub>4</sub>15g/L
 +
 +
NaCl 2.5g/L
 +
 +
NH<sub>4</sub>Cl 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 H<sub>4</sub>O
 +
 +
2. Filter sterilize.
 +
 +
 +
2ml 1mol/L MgSO<sub>4</sub>
 +
 +
1.Dissolve 24.65g MgSO<sub>4</sub>4.7H<sub>2</sub>O in 100ml water.
 +
 +
2. Autoclave to sterilize. 121°C for 20 minutes.
 +
 +
 +
0.1ml 1mol/L CaCl<sub>2</sub>
 +
 +
1.Dissolve 1.11g CaCl<sub>2</sub> in 10ml water.
 +
 +
2. Autoclave to sterilize. 121°C for 20 minutes.
 +
 +
 +
2g  Casamino acids
 +
 +
Dissolve in 1 X M9 salt directly
 +
 +
 +
20ml thiamine
 +
 +
1. Dissolve 0.337 g Casamino acids in 20mL water;
 +
 +
2. Autoclave to sterilize. 121°C for 20 minutes.
 +
 +
Combine above solutions using sterile technique and store at 4°C.
 +
 +
==Standard==
 +
 +
 +
This standard defines the measurement of the PoPS of a specific Biobrick. It defines how to measure  PoPS in a relative unit , not a absolute value.
 +
 +
 +
=== Strains:===
 +
The system should be measured in the strain of Top10.
 +
 +
 +
=== Plasmid:===
 +
The Biobrick parts measured must be supplied in the plasmid pSB1A3.
 +
 +
 +
=== Reporter:===
 +
The Part BBa_I13504 is chosen as the reporter of all the PoPS output of a Biobrick.
 +
 +
 +
=== Medium:===
 +
The cultures should be grown in the M9 medium.
 +
The recipe of M9 medium :
 +
 +
1X  M9 salts:
 +
 +
Na<sub>2</sub>HSO<sub>4</sub> 33.9g/L
 +
 +
KaH<sub>2</sub>SO<sub>4</sub> 15g/L
 +
 +
NaCl 2.5g/L
 +
 +
NH<sub>4</sub>Cl 5g/L
 +
 +
Dissolve in 1L H<sub>2</sub>O
 +
 +
1mM thiamine hydrochloride ;
 +
 +
0.4% glycerol ;
 +
 +
0.2% casamino acids ;
 +
 +
2mM MgSO<sub>4</sub> ;
 +
 +
0.1mM CaCl<sub>2</sub> ;
 +
 +
 +
=== Measurement detail===
 +
 +
a.  Colonies should be picked from a streaked LB plate and grown for 16~18hrs. Growth condition:37℃,200rpm.
 +
 +
b. Dilute the culture 1:100 when measure a constitutive promoter and 1:1000 for a regulatable promoter.
 +
 +
c. The culture should be grown for 3~4hrs before the measurement begin.
 +
 +
d. Measure fluorescence and OD600 in at most 30mins after sampling.
 +
 +
e. For the regulatable promoter , it is necessary to ensure the same response time. The time interval between
 +
 +
the stimulation and measurement should be exactly the same.
 +
 +
f. All the measurement should be made in the linear range of the equipments. It is necessary to test it before
 +
 +
your experiment.
 +
 +
 +
=== Data process:===
 +
 +
a. A  relative fluorescence unit ustc_st.1 is defined to describe the fluorescence intensity independent from the measurement equipment.
 +
 +
b. The part BBa_ K176009 is selected as the fluorescence standard. The fluorescence intensity of BBa_K176009 was measured as the cultures were grown.
 +
 +
c. Define flu,od as the raw data of fluorescence intensity and OD600. Fit flu/od value correspondent to OD600. Take the correspondent value of flu/od to OD600=0.5 ''std'' as the defined standard  1000 units.
 +
 +
d. All the other raw data [[Image:raw.png|50px]] should be converted to standard flu/od value as shown in equation(1):
 +
 +
 +
 +
[[Image:fomu11.jpg|150px]]                              equation(1)

Latest revision as of 02:09, 22 October 2009

USTC
Home Team Project Modeling Parts Standard & Protocol Software Tool Human Practice Notebook

Team:USTC/Standard & Protocol

Contents

Protocol

Assembly 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

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)/M01.00E-061.00E-061.00E-061.00E-061.00E-061.00E-041.00E-041.00E-041.00E-041.00E-04
C(Atc)/ng/ml00220200200002202002000


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 MgSO4

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

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


0.1ml 1mol/L CaCl2

1.Dissolve 1.11g CaCl2 in 10ml water.

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


2g Casamino acids

Dissolve in 1 X M9 salt directly


20ml thiamine

1. Dissolve 0.337 g Casamino acids in 20mL water;

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

Combine above solutions using sterile technique and store at 4°C.

Standard

This standard defines the measurement of the PoPS of a specific Biobrick. It defines how to measure PoPS in a relative unit , not a absolute value.


Strains:

The system should be measured in the strain of Top10.


Plasmid:

The Biobrick parts measured must be supplied in the plasmid pSB1A3.


Reporter:

The Part BBa_I13504 is chosen as the reporter of all the PoPS output of a Biobrick.


Medium:

The cultures should be grown in the M9 medium. The recipe of M9 medium :

1X M9 salts:

Na2HSO4 33.9g/L

KaH2SO4 15g/L

NaCl 2.5g/L

NH4Cl 5g/L

Dissolve in 1L H2O

1mM thiamine hydrochloride ;

0.4% glycerol ;

0.2% casamino acids ;

2mM MgSO4 ;

0.1mM CaCl2 ;


Measurement detail

a. Colonies should be picked from a streaked LB plate and grown for 16~18hrs. Growth condition:37℃,200rpm.

b. Dilute the culture 1:100 when measure a constitutive promoter and 1:1000 for a regulatable promoter.

c. The culture should be grown for 3~4hrs before the measurement begin.

d. Measure fluorescence and OD600 in at most 30mins after sampling.

e. For the regulatable promoter , it is necessary to ensure the same response time. The time interval between

the stimulation and measurement should be exactly the same.

f. All the measurement should be made in the linear range of the equipments. It is necessary to test it before

your experiment.


Data process:

a. A relative fluorescence unit ustc_st.1 is defined to describe the fluorescence intensity independent from the measurement equipment.

b. The part BBa_ K176009 is selected as the fluorescence standard. The fluorescence intensity of BBa_K176009 was measured as the cultures were grown.

c. Define flu,od as the raw data of fluorescence intensity and OD600. Fit flu/od value correspondent to OD600. Take the correspondent value of flu/od to OD600=0.5 std as the defined standard 1000 units.

d. All the other raw data Raw.png should be converted to standard flu/od value as shown in equation(1):


Fomu11.jpg equation(1)

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