Experiments
This section details all the equipment used as well as the steps and protocols for each experiment in each of the three phases of the project. Due to time constraints, some planned and prepared experiments could not be carried out; they are also detailed to demonstrate the thought process behind the entire project.
PHASE I: Cloning Experiments
A. Inventory of Primers and Genes
B. List of Experiments Performed
-
Gene Amplification
- PCR
- Electrophoresis
-
Fragment Purification
- In liquid
- On gel
-
Vector Digestion by Restriction Enzymes
- pBAD-hisC digest with HinIII and NcoI
- pOK12 digest with HindIII and XhoI
-
Cloning
- SLIC
- Gibson
-
Plasmid Insertion into E. coli DH5α
- Super competent bacteria
- Transformation
-
Clone Verification
- Colony PCR
- Restriction profile
- Sequencing
Cloning diagrams
PHASE II: Characterization of the Inducible Promoter and the VIRBAC Receptor
A. Experiments Performed
-
Fluorescence Characterization with Xylose Induction
- Xylose gradient
- Spectrofluorimetry
-
Receptor Production with Xylose Induction
- Xylose gradient
- Western blot
-
Receptor Production with Xylose Induction and Glucose Inhibitor
- Xylose induction
- Glucose gradient
- Western blot
-
Membrane Insertion Verification
- Xylose induction
- Cellular fractionation
- Western blot
B. Planned (Not Performed) Experiments
-
Fluorescence Characterization with Xylose Induction and Glucose Inhibitor
- Xylose induction
- Glucose gradient
- Spectrofluorimetry
-
Membrane Fluorescence
- Spheroplasts
- Immunolabeling
-
Measurement of Receptor Dimerization
- Caffeine gradient
- Spectrofluorimetry
PHASE III: Characterization of the Double Level Expression System METALE
A. Inventory of Primers Used
B. List of Experiments Performed
-
Fluorescence Characterization by Microscopy (Constitutive Promoter)
- Sample preparation
-
Fluorescence Characterization with TECAN (Constitutive Promoter)
- Spectrofluorimetry
-
Mutagenesis of RBS (Constitutive Promoter)
- Quick change PCR reaction
- Transformation
- Screening
- Mini prep
- Sequencing
-
Fluorescence Characterization of Mutants (Constitutive Promoter)
- Spectrofluorimetry
C. Planned (Not Performed) Experiments
-
Fluorescence Characterization by Microscopy (Inducible Promoter)
- Caffeine induction
- Sample preparation
-
Fluorescence Characterization with TECAN (Inducible Promoter)
- Caffeine gradient
- Spectrofluorimetry
-
Mutagenesis of RBS (Inducible Promoter)
- Quick change PCR reaction
- Transformation
- Screening
- Mini prep
- Sequencing
-
Fluorescence Characterization of Mutants (Inducible Promoter)
- Caffeine gradient
- Spectrofluorimetry
-
Verification of Protein Production (Inducible Promoter)
- Caffeine gradient
- Western bloc
-
Visualization of Bacterial Lysis
- Transformation (cytoplasmic fluorescent protein)
- Caffeine gradient
- Video of bacteria