Team:Imperial College London/Wetlab/Protocols/Thermoinduction

From 2009.igem.org

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(New page: =Effect of Temperature= ==Aims== We aim to investigate the behaviour of the pLambda promoter when subjected to a change in temperature. ==Assay== For this assay, we will measure the re...)
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=Effect of Temperature=
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{{Imperial/09/TemplateTop}}
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{{Imperial/09/Tabs/Main/Wetlab/Protocols}}
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=Thermoinduction Assay=
==Aims==
==Aims==
-
We aim to investigate the behaviour of the pLambda promoter when subjected to a change in temperature.
+
*To investigate the behaviour of the Harvard construct and show that when temperature is low (at 28 degrees), there is low fluorescence, while fluorescence increases when temperature is raised to 42 degrees.
==Assay==
==Assay==
-
 
+
Cells will be grown at 28 degrees. After equilibrating, they will be shifted to 42 degrees, and the change in fluorescence will be observed.
-
For this assay, we will measure the response of pLamda promoter to increasing temperatures. We want to monitor the promoter activity to plot a graph of activity against temperature. We will fit this to a hill function to model our pLambda promoter. This reates to M3 since we will be using an increase in temperature to induce M3 and result in the beginning of cell death.
+
==Equipment==
==Equipment==
 +
*Heated water bath
 +
*42 degrees incubator
 +
*Multi-well plate reader
 +
*96 well plates
 +
*15ml falcon tubes
 +
*50ml falcon tubes
-
1) Thermometer
+
==Materials==
 +
*M9 media with 0.2% Casamino acid supplemented with 1mM CaCl2 and 1mM MgSO4
-
2) Heated water bath
+
==Cells==
-
 
+
*Test construct CRP-GFP-Lac-RFP
-
3) Spectrophotometer
+
*Negative I13504 RBS-GFP-TT
-
 
+
*Positive I13522 Ptet-RBS-GFP-TT
-
4) Fluorimeter
+
-
 
+
-
5) 96 well plates
+
-
 
+
-
==Reagents==
+
-
 
+
-
1) tetracycline, 10 μg ml−1
+
-
 
+
-
2) ampicillin, 100 μg ml−1
+
-
 
+
-
3) 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (XGal), 0.04 mg ml−1.
+
-
 
+
-
4) LB broth
+
==Protocol==
==Protocol==
-
1)  Mix:
 
-
*tetracycline, 50 μg
 
-
*ampicillin, 500 μg
 
-
*5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (XGal), 0.20 mg
 
-
*5mL of LB (Luria-broth) and plate e. coli. Grow overnight at 28 °C.
 
-
*M9 medium
 
 +
===Day 1 evening===
 +
1) Using a loop, pick out a single colony of the relevant cells from plates (in cold room)/<br>
 +
Or inoculate with starter culture 1 in 50.  <br>
 +
Innoculate the  cells on M9 + 0.5% Glucose and grow them overnight at 37 °C<br>
 +
<br>
 +
2)Label the 15ml falcon tubes as following: <br>
 +
<br>
 +
Tube 1: 28 degrees blank<br>
 +
Tube 2: 28 degrees Harvard-GFP construct<br>
 +
Tube 3: 28 degrees negative<br>
 +
Tube 4: 28 degrees positive<br>
 +
Tube 5: shift from 28 to 42 degrees blank<br>
 +
Tube 6: shift from 28 to 42 degrees Harvard-GFP construct<br>
 +
Tube 7: shift from 28 to 42 degrees negative<br>
 +
Tube 8: shift from 28 to 42 degrees positive<br>
 +
Tube 9: 42 degrees blank<br>
 +
Tube 10: 42 degrees Harvard-GFP construct<br>
 +
Tube 11: 42 degrees negative<br>
 +
Tube 12: 42 degrees positive<br>
 +
<br>
 +
Label another set of tubes similarly. <br>
 +
<br>
 +
3) Prepare 80ml of M9 media + 0.5% glucose from stock glucose solution<br>
 +
For 30% glucose stock, dilution factor = 30/0.5= 60x<br>
 +
Therefore, vol of glucose solution = (vol of M9 media)/60<br>
 +
<br>
 +
===Day 2===
 +
1) Calibrate the multi-well plate reader with the I13522 overnight cells<br>
<br>
<br>
-
 
+
High gain= 50%<br>
-
2) We need to ensure that bacterial cultures are at mid-log growth phase, by measuring the optical density at 600 nm (OD600).
+
Low gain = 6% (no need to change) <br>
-
 
+
<br>
-
3) Check if cell count shows that bacterial is growing at mid-log phase.
+
2)The previously mentioned tubes are filled with 3ml of M9 + 0.5% glc<br>
-
 
+
<br>
-
4) If mid-log phase is reached, transfer cultures to water baths at 28 °C, 30°C, 36°C, 39°C, 42°C, respectively, with both a control and a transformed culture <br>
+
3)The overnight culture is diluted 1 in 20 into the first set of the newly prepared 15ml tubes<br>
-
 
+
<br>
-
5)  OD600 and GFP/beta-galactosidase readings (refer to Assays) should be taken at half hourly intervals. <br>
+
4) The tubes with cells are grown in the following conditions: <br>
-
 
+
<br>
-
 
+
The 28 degrees cells and shift cells : at 28 degrees, <br>
-
===Inoculation===
+
42 degrees cells : at 42 degrees<br>
-
1. Three 5 ml cultures of M9 minimal medium5 supplemented with 0.2% casamino acids and 1mM Thiamine Hydrochloride (supplemented M9 medium) and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of MG1655 containing BBa_T9002. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of MG1655 containing a BBa_T9002 mutant lacking a GFP expression device (T9002m) described in the stability section. <br>
+
<br>
-
 
+
5)The water bath is switched on to 55 degrees<br>
-
 
+
<br>
-
2. Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm. <br>
+
6) For the second set of tubes (without cells)<br>
-
 
+
<br>
-
===Growing up cells===
+
28 degrees tubes are put in the 28 degrees incubator<br>
-
 
+
42 degrees tubes in the 42 degrees incubator<br>
-
3. Cultures were diluted 1:1000 into 5.5 ml of pre-warmed fresh medium and grown to an OD600 of 0.15 under the same conditions as before (this growth took 4.5 hrs on average). <br>
+
shift tubes (w/o cells) are placed in the 55 degrees water bath. <br>
-
 
+
<br>
-
 
+
7) Subsequent OD and GFP readings are taken using the following settings: <br>
-
===Measurement===
+
<br>
-
 
+
Layout for the protocols can be imported as HVD-GFP<br>
-
4. Six 200 µl aliquots of each of the cultures were transferred into a flat-
+
Measure: IPTG_GFP_FL (low gain) <br>
-
bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br>
+
Script: HVD-GFPhigain (high gain) <br>
-
 
+
<br>
-
 
+
8) We need to ensure that bacterial cultures are at mid-log growth phase. <br>
-
5. Three wells were each filled with 200 Il of medium to measure the absorbance background. <br>
+
<br>
-
 
+
9) When mid-log phase is reached, an OD and GFP reading is taken of the cells. The tubes with cells are then added to the second set of tubes, and a subsequent OD and GFP reading is taken (time=0) <br>
-
 
+
<br>
-
6. The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 sec counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 sec, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed, 5 sec). Time between repeated measurements was 69 sec. Cells appear to grow exponentially for the duration of the plate reader measurement protocol
+
10) The second set of tubes (now with cells) are now grown at the following conditions: <br>
-
 
+
<br>
-
 
+
28 degree tubes: at 28 degrees<br>
-
5. After 10 min in the multi-well fluorimeter, inducer was added to three of the wells for each culture to a final concentration of 1E-7 M.
+
Shift and 42 degree tubes: at 42 degrees<br>
-
 
+
<br>
-
6. The plate was again incubated in the multi-well fluorimeter and assayed as before.
+
11) The cells are now sampled for their GFP and OD readings half hourly for around 5 hours until the GFP fluorescence plateaus off. <br>
-
'''Bold text'''
+
<br>
-
 
+
{{Imperial/09/TemplateBottom}}
-
==Wetlab processing==
+
-
 
+
-
A graph of temperature against cell count would be plotted to access the viability of e. coli with regards to temperature.
+
-
 
+
-
==References==
+
-
 
+
-
<biblio> >#hot pmid=15652426 </biblio>
+
-
 
+
-
 
+
-
=Dynamic Response=
+
-
 
+
-
==Equipment==
+
-
 
+
-
* Spectrophotometer
+
-
* Fluorimeter
+
-
* 96 well plates
+
-
 
+
-
 
+
-
==Reagents==
+
-
 
+
-
* M9 medium
+
-
 
+
-
==Protocol==
+
-
 
+
-
===Inoculation===
+
-
1. Three 5 ml cultures of M9 minimal medium5 supplemented with 0.2% casamino acids and 1mM Thiamine Hydrochloride (supplemented M9 medium) and antibiotic (kanamycin, 20 µg/ml) were inoculated with single colonies (~2mm ø) from a freshly streaked plate of MG1655 containing BBa_T9002. One 5 ml culture was inoculated with a single colony from a freshly streaked plate of MG1655 containing a BBa_T9002 mutant lacking a GFP expression device (T9002m) described in the stability section. <br>
+
-
 
+
-
 
+
-
2. Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm. <br>
+
-
 
+
-
===Growing up cells===
+
-
 
+
-
3. Cultures were diluted 1:1000 into 5.5 ml of pre-warmed fresh medium and grown to an OD600 of 0.15 under the same conditions as before (this growth took 4.5 hrs on average). <br>
+
-
 
+
-
 
+
-
===Measurement===
+
-
 
+
-
4. Six 200 µl aliquots of each of the cultures were transferred into a flat-
+
-
bottomed 96 well plate (Cellstar Uclear bottom, cat. # T-3026-16, Greiner). <br>
+
-
 
+
-
 
+
-
5. Three wells were each filled with 200 Il of medium to measure the absorbance background. <br>
+
-
 
+
-
 
+
-
6. The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 sec counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 sec, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed, 5 sec). Time between repeated measurements was 69 sec. Cells appear to grow exponentially for the duration of the plate reader measurement protocol
+
-
 
+
-
 
+
-
5. After 10 min in the multi-well fluorimeter, inducer was added to three of the wells for each culture to a final concentration of 1E-7 M.
+
-
 
+
-
6. The plate was again incubated in the multi-well fluorimeter and assayed as before.
+
-
'''Bold text'''
+

Latest revision as of 18:16, 15 October 2009



Contents

Thermoinduction Assay

Aims

  • To investigate the behaviour of the Harvard construct and show that when temperature is low (at 28 degrees), there is low fluorescence, while fluorescence increases when temperature is raised to 42 degrees.

Assay

Cells will be grown at 28 degrees. After equilibrating, they will be shifted to 42 degrees, and the change in fluorescence will be observed.

Equipment

  • Heated water bath
  • 42 degrees incubator
  • Multi-well plate reader
  • 96 well plates
  • 15ml falcon tubes
  • 50ml falcon tubes

Materials

  • M9 media with 0.2% Casamino acid supplemented with 1mM CaCl2 and 1mM MgSO4

Cells

  • Test construct CRP-GFP-Lac-RFP
  • Negative I13504 RBS-GFP-TT
  • Positive I13522 Ptet-RBS-GFP-TT

Protocol

Day 1 evening

1) Using a loop, pick out a single colony of the relevant cells from plates (in cold room)/
Or inoculate with starter culture 1 in 50.
Innoculate the cells on M9 + 0.5% Glucose and grow them overnight at 37 °C

2)Label the 15ml falcon tubes as following:

Tube 1: 28 degrees blank
Tube 2: 28 degrees Harvard-GFP construct
Tube 3: 28 degrees negative
Tube 4: 28 degrees positive
Tube 5: shift from 28 to 42 degrees blank
Tube 6: shift from 28 to 42 degrees Harvard-GFP construct
Tube 7: shift from 28 to 42 degrees negative
Tube 8: shift from 28 to 42 degrees positive
Tube 9: 42 degrees blank
Tube 10: 42 degrees Harvard-GFP construct
Tube 11: 42 degrees negative
Tube 12: 42 degrees positive

Label another set of tubes similarly.

3) Prepare 80ml of M9 media + 0.5% glucose from stock glucose solution
For 30% glucose stock, dilution factor = 30/0.5= 60x
Therefore, vol of glucose solution = (vol of M9 media)/60

Day 2

1) Calibrate the multi-well plate reader with the I13522 overnight cells

High gain= 50%
Low gain = 6% (no need to change)

2)The previously mentioned tubes are filled with 3ml of M9 + 0.5% glc

3)The overnight culture is diluted 1 in 20 into the first set of the newly prepared 15ml tubes

4) The tubes with cells are grown in the following conditions:

The 28 degrees cells and shift cells : at 28 degrees,
42 degrees cells : at 42 degrees

5)The water bath is switched on to 55 degrees

6) For the second set of tubes (without cells)

28 degrees tubes are put in the 28 degrees incubator
42 degrees tubes in the 42 degrees incubator
shift tubes (w/o cells) are placed in the 55 degrees water bath.

7) Subsequent OD and GFP readings are taken using the following settings:

Layout for the protocols can be imported as HVD-GFP
Measure: IPTG_GFP_FL (low gain)
Script: HVD-GFPhigain (high gain)

8) We need to ensure that bacterial cultures are at mid-log growth phase.

9) When mid-log phase is reached, an OD and GFP reading is taken of the cells. The tubes with cells are then added to the second set of tubes, and a subsequent OD and GFP reading is taken (time=0)

10) The second set of tubes (now with cells) are now grown at the following conditions:

28 degree tubes: at 28 degrees
Shift and 42 degree tubes: at 42 degrees

11) The cells are now sampled for their GFP and OD readings half hourly for around 5 hours until the GFP fluorescence plateaus off.

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Retrieved from "http://2009.igem.org/Team:Imperial_College_London/Wetlab/Protocols/Thermoinduction"