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Team:Imperial College London/Wetlab/Protocols/Thermoinduction
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Contents |
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 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
1) Thermometer
2) Heated water bath
3) Spectrophotometer
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
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
2) We need to ensure that bacterial cultures are at mid-log growth phase, by measuring the optical density at 600 nm (OD600).
3) Check if cell count shows that bacterial is growing at mid-log phase.
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
5) OD600 and GFP/beta-galactosidase readings (refer to Assays) should be taken at half hourly intervals.
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.
2. Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
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).
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).
5. Three wells were each filled with 200 Il of medium to measure the absorbance background.
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
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.
2. Cultures were grown in 17 mm test tubes for 15 hrs at 37°C with shaking at 70 rpm.
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).
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).
5. Three wells were each filled with 200 Il of medium to measure the absorbance background.
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
