Protocols

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: Making 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
• CC13: Genome Prep for Restriction Assay

Calibrations

• CA1: Optical Density Calibration
• GFP fluorescence calibration
  • CA2: External GFP fluorescence
  • CA3: Intracellular GFP fluorescence

Promoter Characterisation

• PP1: Lac Promoter Characterisation

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

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