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Team:Imperial College London/Wetlab/Protocols
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[[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth| See the protocol for more details]] | [[Team:Imperial_College_London/Wetlab/Protocols/IPTGgrowth| See the protocol for more details]] | ||
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==Lac characterisation and IPTG effect on protein production== | ==Lac characterisation and IPTG effect on protein production== | ||
Revision as of 11:42, 18 September 2009
- Back to Hub
- Cloning Strategy
- Protocols
- BioBricks
- Results
- Notebook
Contents |
Protocols
- Calibrations
- Top 10 Growth Calibration
- Promoter Characterisation
- Lac Promoter
- Auto Induction
- Protein Production
- Colanic Acid Encapsulation
- Trehalose Production
- Thermoinduction
- Genome Restriction
| Section | Assay | Overview and Aims |
|---|---|---|
| 1.Calibration | TOP10 Growth | * To produce a calibration curve to aid in the normalising of absorbance values. The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10.
|
| 1.Promoter Characterisation | Blah | To find |
| 2.Auto-Induction | Secondary Carbon Source Diauxie Growth |
|
| 2.Auto-Induction | Glucose Time Delay | * Characterise the tunable time duration it takes before GFP expression (M2 activation) |
| 3.Protein Production | IPTG Toxicity | To investigate the effect of our IPTG inducer on growth of our cultures.
|
| 3.Protein Production | Cellulase | Aims |
| 3.Protein Production | PAH | Aims |
| 4.Colanic Acid Encapsulation | Colanic Acid | Colanic acid biosynthesis is both time consuming and metabolically expensive. If the E.ncapsulator is to be used in an industrial setting, then colanic acid mediated protection must be highly efficient. Protective efficiency can be defined as the percentage increase in fluorescence per µl of colanic acid produced (when compared to control cells). The following assay can be used to elucidate this parameter. |
| 8.Genome Restriction | [|In Vitro Restriction] | * By running restriction digests on the genome of E.coli strains, we can investigate the efficiency of our restriction enzyme, taqI and dpnII, on genome deletion.
|
Promoter characterisation assays
Absorbance calibration
Aim
- To produce a calibration curve to aid in the normalising of absorbance values. The relation of absorbance reading to number of cells varies with different cell strains. We are therefore doing one for Top-10.
Assay
Cultures of the E.coli with the relevant vector are grown to various cell densities. A sample of these cultures are taken and a dilution plate is carried out to work out approximate colony forming units per ml of culture. This data set is then combined with absorbance readings to create a graph relating the number of colony forming cells per ml to their absorbance measurements.
This curve then allows us to convert absorbance of a known volume of culture to colony forming units within the culture sample.
See the protocol for more details
Autoinduction assays
Lac characterisation and IPTG effect on protein production
Aims
- Characterise Lac promoter by varying amounts of IPTG
- Determine the IPTG concentration that allows for maximal protein production while still being non-toxic to the cell
Assay
The cells will be grown until OD=0.7. Now, IPTG of various concentrations will be added, and the RFP output will be measured.
The experiment will generate OD and fluoresence data for RFP
The secondary carbon source will be taken from the previous experiment (see Secondary carbon source selection)
The glucose concentration will be taken from commercial autoinduction media (0.05%) – takes about 7 hours to exhaust
See the protocol for more details
