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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
CC9: Preparation of XL1-Blue electrocompetent cells & XL1-Blue quality control
CC10: PCR
CC11: SLIC
CC12: Biobricking parts from oligios

Calibrations

CA1: Optical Density 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	Characterise Lac promoter by varying concentrations of IPTG Determine the IPTG concentration that allows for maximal protein production while still being non-toxic to the cell
3.Polypeptide Production	IPTG Toxicity	To investigate the effect of our IPTG inducer on growth of our cultures. Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined Determine the effect of IPTG toxicity on growth w/o any protein production complications
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	Genome Prep	* Preperation of the genomic DNA for restriction digest assay.
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. Run parallel digests with our chassis of choice (TOP10) and a Dam negative control (of which the DNA should not be methylated) to investigate the effects of methylation on cleavage.

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