Aim: Extract and amplify our genomic UAP1 sequence from yeast DNA by Polymerase chain reaction (PCR).
Protocol: Gene PCR. We ran a 0.8% agarose gel electrophoresis to check our PCR product size.
Conclusion: We successfully amplified the UAP1 gene using PCR (Figure 1 showing a and of the size 1500bp). We can assemble our gene with a digested vector and transform it into chemically competent E. coli cells.
Aim: Linearizing and amplifying our vector IBA3 by Polymerase chain reaction (PCR).
Protocol: Vector PCR. We ran a 0.8% agarose gel electrophoresis to check our PCR product size.
Conclusion: We successfully amplified IBA3 vector using PCR (Figure 1 shows a band size 3215bp). We can now digest the vector with Dpn I enzyme.
Result:
Aim: : Linearization of the IBA3 plasmid for subsequent gene assembly.
Protocol: Plasmid digestion.
Conclusion: Ready plasmid for genomic assembly.
Aim: Assemble our gene UAP1 with digested vector IBA3 and sequentially transform into competenc DHalpha E. coli cells.
Protocol: Gene assembly.
Conclusion: Ready plasmid with recombinant DNA for transformation.
Aim: Transform our cloned vector into the competent cells, DH-alpha E. coli, heat-shock and plate them on LB plate with ampicillin.
Protocol: Transformation.
Conclusion: Four colonies grew on the plate, however, have to verify the correct assembly of the cloned DNA construct by doing a colony PCR.
Mix the competent cells DH-alpha E. coli into the genes assembly and heat-shock them using thermocycler (-17 min).
Aim: Pick the colonies that survived the ampicillin treatment and grow them in liquid cultures to amplify our plasmid DNA.
Protocol: Batch of 5 mL E. Coli from agar plate.
Result: Turbid tubes which indicate bacterial growth.
Conclusion: Sufficient bacterial culture for subsequent experiments such as plasmid purification and protein induction.
Aim: To screen for genetically transformed and verify the assembly of the DNA construct.
Protocol: Colony PCR.
Result: Confirmation of expected size of amplicons.
Conclusion: Assembly and transformation of the UAP1 gene into the IBA3 vector into DHalpha competent cells were successful in 3 of the 4 colonies.
Aim: Isolate plasmid DNA from other DNA.
Protocol: Miniprep.
Result: Sufficient amount of plasmid DNA in 3 cultures, determined by nanodrop.
# | Concentration (ng/µL) |
---|---|
I1 | 199 |
I2 | 164 |
I3 | 99 |
Conclusion: Ready plasmid DNA for sequencing.
Aim: Prove successful insert in vector.
Protocol: 6 tubes for 3 isolated plasmid samples were prepared. 3 tubes were prepared with 5 mM forward primer, and 3 with 5 mM reverse primer. 80-100 ng/µL plasmid DNA was added to each tube, one sample in both forward and reverse tube, then topped to 10 µL total in water. Sent for sanger sequencing to Eurofins Genomics.
Result: Successful insert of plasmid in all three cultures.
Conclusion: Undisputed proof that the target gene is inserted in the bacteria.
Aim: Using AHTc to induce the expression of UPA1 gene.
Protocol: 10 mL bacterial culture is transferred to new flask, a dilution is prepared until 10 µL diluted AHTc is added to each.
Result: Colonies that produce the target protein.
Conclusion: Bacterial cultures in both induced and uninduced states are to be used for the analysis of protein to see if UAP1 is expressed as expected.
Aim: Lyse the cells without using detergent to preserve the protein function.
Protocol: Pelleted with 3 000 rcf for 8 min. Lysis buffer added to 1/10 original volume (1 mL). Lysis buffer consistent with 200 mM, 0.5 mg/mL lysozyme, and 10 mM Tris. Bead lysis for ~40 seconds. Lysate separated into new tubes, then stored on ice for processing.
Conclusion: To determine the effectiveness of the cell lysis, functional assay and sds required.
Aim: Determining the amount of protein by calculation against BSA from induced and non-induced lysed bacterial cultures.
Protocol: Bradford assay.
Result:
Estimated concentration using 5th degree polynomial regression:
Induced (+; µg/µL) | Non-induced (-; µg/µL) |
---|---|
1,368856 | 3,81238 |
1,528336 | 3,651112 |
1,164268 | 3,804776 |
0,479516 | 3,863736 |
Conclusion: Determined the concentration of the protein solutions in the different cultures.
Aim: Validate protein expression by the expected size.
Protocol: SDS-PAGE.
Result: Confirmation of expected size of protein.
Conclusion: Successful expression in induced vs non-induced.
Aim: Quantitative measurement of UAP1 activity.
Protocol: Dephosphorylation assay.
Result:
Conclusion: Unable to confirm increased activity caused by UAP1 in induced, versus non-induced culture.