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Team:Imperial College London/Wetlab/Protocols
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* <b>CC6</b>: Transformation into Top10 using chemical competence | * <b>CC6</b>: Transformation into Top10 using chemical competence | ||
* <b>CC7</b>: Make LB plates | * <b>CC7</b>: Make LB plates | ||
| + | * <b>CC8</b>: Extracting DNA from the registry | ||
==[[Team:Imperial College London/Wetlab/Protocols/Calibration| Calibrations]]== | ==[[Team:Imperial College London/Wetlab/Protocols/Calibration| Calibrations]]== | ||
Revision as of 16:14, 24 September 2009
Contents |
Protocols
- Back to Hub
- Cloning Strategy
- Protocols
- BioBricks
- Results
- Notebook
Cell culturing protocols
- CC1: Miniprep
- CC2: Midiprep
- CC3: Making cells competent
- CC4: Ligation
- CC5: Transformation into BL21 using electrical shock
- CC6: Transformation into Top10 using chemical competence
- CC7: Make LB plates
- CC8: Extracting DNA from the registry
Calibrations
- CA1: OD calibration
- GFP fluorescence calibration
- CA2: External GFP fluorescence
- CA3: Intracellular GFP fluorescence
Autoinduction
- AI1: Secondary Carbon Source Experiment
- AI2: Glucose Time delay
Protein Production
- PP1: IPTG Toxicity
- PP2: IPTG characterisation
- PP3: Cellulase
- PP4: PAH
Encapsulation
- EN1: Colanic acid production amount
- EN2: Colanic acid protection from pH
- EN3: Trehalose production
Killing
- KI1: Thermoinduction
- KI2: Restriction Enzyme activity
| Section | Assay | Overview and Aims |
|---|---|---|
| 1.Calibration Curves | 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 | Glucose Time Delay | * Characterise the tunable time duration it takes before GFP expression (M2 activation) |
| 2.Auto-Induction | IPTG |
|
| 3.Polypeptide Production | IPTG Toxicity | To investigate the effect of our IPTG inducer on growth of our cultures.
|
| 3.Polypeptide Production | Cellulase | Aims |
| 3.Polypeptide Production | PAH | Aims |
| 5.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. |
| 6.Trehalose | Colanic Acid | asddaf |
| 7.Thermoinduction | Thermoinduction | ghj |
| 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.
|
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
