Notebook
Overview
All of our lab work were documented using Benchling. We've separated our lab work into cloning work and proteins expression & purification. Click on the buttons on the right side to toggle the lab notes for the respective categories.
Week 1
15 April - 21 April
Protein Expression & Purification
- Overnight culture of alpha and beta prefoldins with catcher.
- Expression of prefoldins by IPTG induction in T7 express cells.
- Bug buster and SDS-PAGE gel run to check for protein size.
- Gel showed beta prefoldin as a strong band at the correct size however alpha was too small and possibly did not contain the fused catcher protein.
Week 2
22 April - 28 April
Protein Expression & Purification
- Overnight culture of alpha and beta prefoldins with catcher.
- Expression of prefoldins by IPTG induction in T7 express cells.
- Freezing of samples
Week 8
3 June - 9 June
Cloning
- PC2 lab induction.
- Construct lab plan, compile protocols and reagent list.
Protein Expression & Purification
- Lab induction
- Lab plan written
- Ordered materials
Week 9
10 June - 16 June
Cloning
- Design and order G-blocks for DBAT, Duke's DBAT, LXYL-P1-2, TycA and PAM enzymes from IDT.
Protein Expression & Purification
- Meeting with supervisor to discuss lab plan
Week 10
17 June - 23 June
Cloning
- Preparation of Luria Broth Agar with Ampicillin plates.
- Small scale grow up of pET19b plasmid containing competent Dh5-alpha E.coli cells.
- pET19b plasmid extracted and purified using Promega miniprep kit, plasmid concentration and purity quantified by nanodrop.
Protein Expression & Purification
- Building induction/ OGTR Induction.
Week 11
24 June - 30 June
DNA
- Calculate PCR thermocycling conditions and mastermix contents.
Protein Expression & Purification
- Bug-buster and SDS-PAGE of samples prepared in week 2 (α-PFD + Catcher before and after IPTG induction, β-PFD + Catcher before and after IPTG induction).
- SDS-PAGE gels showed expression of all proteins was achieved in the soluble fraction however there was a low expression of alpha prefoldin, and beta-prefoldin was found in the insoluble fraction.
- Following successful expression results, cells were lysed via sonication by Nga, spun down and separated into soluble and insoluble fractions of proteins.
- Soluble and insoluble protein was prepared and incubated onto Ni-NTA beads (soluble α+β, insoluble α, insoluble β) then loaded onto gravity columns for elution by buffers with different imidazole concentrations into fractions.
- Elution fractions were run on SDS-PAGE to show presence of more pure protein.
- Made buffers for Ni-NTA Histrap protein purification (binding and elution buffers) and insoluble fraction denaturing (triton 1X and guanidine denaturing buffer).
- Made PC2 labels for chemicals.
Week 12
1 July - 7 July
Cloning
- Linearise pET19b backbone by PCR amplification (Phusion DNA polymerase). The concentration and purity quantified by nanodrop, then visualised by agarose gel electrophorepsis.
- Rerun PCR of pET19b backbone with optimised annealing temperature.
- Circular plasmid DNA removed by DpnI digest (NEB), purified using QIAGEN PCR clean up kit.
- Gibson assembly of G-blocks into pET19b backbone, concentration and purity of DNA quanitified by nanodrop.
- Transformation of assembled constructs into competent T7 Express E.coli (NEB), then plated onto selection plates with Ampicillin and grown overnight.
Protein Expression & Purification
- Prepare Bolt MES buffer and protein fixative buffer (25% isopropanol, 10% acetic acid) for PAGE gels .
- Prepared elution buffer concentration gradient solutions for His-tag protein purification.
- Starter culture of prefoldins (α-PFD, α-PFD + Catcher, β-PFD, β-PFD + Catcher) in 10ml Luria broth overnight with ampicillin.
- Addition of starter cultures into separate autoclaved LB broths (1 L) with ampicillin at 1 X concentration with incubation at 37°C, 200 rpm (for fast growing) until 0.6 OD is reached.
- Cultures were induced with IPTG and incubated at 20°C, 200 rpm overnight (for protein production).
- Bug buster of induced and non-induced (control) samples of alpha, alpha +C, beta, beta +C, followed by SDS-PAGE to show protein production in response to IPTG induction and presence of proteins in soluble fractions (apart from beta and beta plus catcher which appeared in the insoluble fraction).
- Spin down culture to form pellets and freeze in -40°C.
- Filter and degas solvents to be used for AKTA in week 13.
- Training for AKTA start (chromatography machine for protein purification) and sonicator (for cell lysis).
WEEK 13
8 July - 14 July
Cloning
- Gibson assembly of TycA into pET19b, troubleshooting molar ratio, transformed into T7 Express E.coli and grown overnight.
- Small scale grow up of DBAT, Duke's DBAT, PAM, LYXL and TycA.
- Extraction and purification of plasmids by miniprep (QIAGEN), quantified by nanodrop.
Protein Expression & Purification
- Cell lysis by sonication of pellet samples prepared in week 12
- Purify alpha +C, beta +C and alpha via AKTA start machine
- SDS-PAGE of fractions which showed clean proteins for all samples without contaminants however lots of protein was found in flow though (did not originally bind well to column)
- Concentration and buffer exchange of alpha +C, beta + C and alpha however beta + C precipitated in the concentration tube (may need to use dialysis for buffer exchange instead)
- BCA assay on beta +C, alpha w/ and without catcher to determine protein concentrations
- Reload alpha +C and beta +C flow-through on AKTA to collect protein found in original flow-through
- SDS page found flow through of second AKTA run still contained a lot of his-tagged protein not bound to column.
Week 14
15 July - 21 July
Cloning
- Plasmid DNA concentrated using Promega DNA clean up Kit.
- Small scale grow up, miniprep (QIAGEN) of TycA, PAM, Duke's DBAT and LXYL containing E.coli strains.
- Samples submitted for sequencing by Ramaciotti Centre for Genomics.
Week 15
22 July - 28 July
Cloning
- Replated DBAT, PAM, TycA and LXYL strains.
- Ordered S. cerevisiae codon optimised LXYL-P1-2 gBlock from IDT.
- Compiled reagent list and protocols for transformation of LXYL-P1-2 into S. cerevisiae.
- Preparation of YEPD media and agar plates.
- Obtained S. cerevisiae BY4741 strain, courtesy of Marc Wilkin's Laboratory.
Protein Expression & Purification
- Run alpha flow-through on AKTA to collect protein found in original flow-through.
- Lyse E.coli cells containing beta prefoldin (from week 12 pellet samples) via sonication, and incubate washed Ni-NTA resin with supernatant (soluble protein).
- Wash resin and elute beta prefoldin from gravity column.
- SDS-PAGE, found beta prefoldin protein in fractions with limited contamination from other proteins.
- Concentration of alpha +C, alpha, beta +C and beta samples and flow-throughs.
- BCA assay for all four prefoldins to determine protein concentrations.
Week 16
29 July - 4 August
Cloning
- Colony PCR of DBAT, LXYL, TycA, PAM and Duke's DBAT, amplified sequences visualised by gel electrophoresis.
- Designed primers to add "TAA" stop codon by PCR-directed codon addition of Duke's DBAT, primers ordered from Sigma Aldrich.
Week 18
12 August - 18 August
Cloning
- PCR-directed codon addition of Duke's DBAT, visualisation of PCR product by gel electrophoresis and quantified by nanodrop.
- PCR-directed codon addition repeated with changes to annealing temperature and extension time.
- Ligation of LXYL S. cerevisiae pD1204 construct by Golden gate assembly and transformed into T7 Express E.coli.
- Gibson Assembly of Duke's DBAT, TycA, and LXYL into pET19b, pET19b and LXYL+pD1204 constructs transformed into T7 Express E. coli.
- The following was performed on the transformants: Small scale grow up, plasmid DNA extracted (QIAGEN miniprep kit), submitted for sequencing (Ramaciotti Centre for Genomics), colony PCR, gel electrophoresis and glycerol stocks for long term storage.
Protein Expression & Purification
- Starter cultures of alpha and beta prefoldins with and without catcher.
- Large scale grow-up and IPTG induction.
- Bug buster and SDS-PAGE found all samples contained protein but their was slightly less beta prefoldin protein than others.
- Pellets were harvested and stored in the -40°C freezer.
Week 19
19 August - 25 August
Cloning
- Sequence alignment of constructs, unsuccessful assembly.
- Ordered Duke's DBAT gBlock with stop codon from IDT.
Week 20
26 August - 1 September
Cloning
- Gibson assembly of Duke's DBAT and pET19b, construct transformed into T7 Express E. coli.
- DBAT, PAM, LXYL(e) and TycA plasmids submitted for sequencing. Insert amplified by PCR and visualised by gel electrophoresis.
- Colony PCR of LXYL(s), visualised by gel electrophoresis.
- Small scale grow up, miniprep and glycerol stocks of Duke's DBAT, LXYL(e), LXYL(s) and TycA.
- Research on possible assays for characterisation ATF1.
Protein Expression & Purification
- Purification of alpha +C, beta +C and alpha using AKTA start machine.
- Concentration of alpha +C, alpha, beta +C and beta samples and flow-throughs.
- Starter cultures of beta, PAM and DBAT prepared.
- Large scale grow-up and IPTG induction.
- PAM and DBAT pellets were harvested and stored in -40°C freezer.
Week 21
2 September - 8 September
Cloning
- Colony PCR of LXYL(e), Duke's DBAT and TycA under optimal annealing temperatures and extension times
- Golden gate assembly of LXYL-P1-2 into pD1204 (provided by Joshua Hamey) and transformation into T7 Express E. coli
- Grow up of U, T, L(e), L(y), miniprep and samples submission for sequencing (Ramaciotti Centre)
- Gibson assembly of TycA+pET19b with time, concentration and gibson master mix controls. Transformed into T7 Express E. coli
- LXYL(e) gibson assembly and transformation
- Grow up of TycA and Duke DBAT and miniprep
- Prepared LB Agar and Chloramphenicol plates
- Transformed linear pSB1C3 and RFP+pSB1C3 into competent HB101 E.coli and plated
- Restreaked pink colonies on new plates to test plate batches
Protein Expression & Purification
- BCA assay for all four prefoldins to determine protein concentrations.
- Bug buster of PAM and DBAT showed good beta expression. However, lots of PAM in the insoluble fraction and no DBAT expression could be seen.
- Native page was performed on prefoldins to demonstrate assembly of assemblase hexameric scaffold. Successful results of assembly were achieved.
- Gravity column purification of PAM and DBAT yielded no soluble protein thus purification was unsuccessful.
- Purification of beta via AKTA start machine.
Week 22
9 September - 15 September
Cloning
- Sequence alignment.
- Submit TycA#3 and Duke#1 for sequencing (Ramaciotti).
- LXYL(e) gibson assembly and transformation.
- Resuspended and transformed ATF1 (alcohol acetyltransferase) into HB101 E.coli, plated and grown overnight.
- Remade LB Agar + CAM plates.
- Troubleshoot Agar plates with RFP and linear controls and transformed into T7 Express E. coli.
- Troubleshoot CAM plates with known CAM resistant strains.
- Resuspended Red Luciferase and EPIC Luciferase DNA and transformed into T7 Express E.coli.
- Grow up of ATF, Red Luciferase and EPIC Luciferase.
- Colony PCR and miniprep of ATF, Red Luciferase and EPIC Luciferase. Visualised by gel electrophoresis.
Protein Expression & Purification
- Expression conditions were tested (IPTG and temperature) for PAM and DBAT.
- Bug buster of the different PAM and DBAT cultures were still insoluble.
- Purification by AKTA for PAM and DBAT samples show no protein binding and elution from the column.
- The Ellman’s assay was tested and a standard curve as constructed.
Week 23
16 September - 22 September
Cloning
- Gel of TycA, Duke’s DBAT, LXYL+pET19b and LXYL+pD1204.
- Restriction enzyme digest of TycA, Duke’s DBAT, LXYL+pET19b, DBAT, PAM. Visualised by gel electrophoresis.
- Colony PCR of ATF colonies, imaged by gel electrophoresis.
- DpnI digest and PCR clean up of ATF4, ATF28 and ATF29 PCR products.
- Gibson assembly of ATF4, ATF28, ATF29 colonies and transformation into T7 Express cells.
- Colony PCR of ATF28/29, visualised by gel electrophoresis.
- Concentrate PAM and DBAT fractions and run on gel.
Protein Expression & Purification
- A 1L expression of alpha +C and beta +C was performed.
- More expression conditions for PAM and DBAT were tested.
- Bug buster shows expression of insoluble PAM while DBAT cultures contain both soluble and insoluble protein.
- SDS-PAGE of cell free lysate and soluble fraction of cultures reconfirms findings.
- Purification of alpha+C and beta+C as well as one PAM and one DBAT sample on AKTA.
- Buffer exchange of alpha+C and beta+C via dialysis.
- Concentration of PAM and DBAT fractions were run on gel showing very light PAM and DBAT bands.
Week 24
23 September - 29 September
cloning
- Colony PCR of mCerulean3, TycA and ATF1 #28.
- DpnI digest (NEB) and DNA clean up (QIAGEN) of mCerulean3 and ATF1 fragments.
- Gibson assembly of mCerulean3, TycA, LXYL, Duke's DBAT and ATF1 into pET19b and transformed into T7 Express E.coli.
- Colony PCR of mCerulean3, TycA, LXYL, Duke's DBAT and ATF1. DNA fragments visualised by gel electrophoresis on a 1% agarose gel.
- Small scale grow up and miniprep of positive colonies.
Protein Expression & Purification
- Purified and non-purified PAM and DBAT bands were excised from SDS-PAGE gel and sent for mass spectrometry analysis. Results show both PAM and DBAT are present in the soluble fractions however PAM is at a very low concnetration.
- Expression of 1 L PAM cultures (0mM IPTG and 0.1 mM IPTG).
- Bug buster performed on PAM cultures. Both showed PAM in insoluble fraction.
- PAM (0.1 mM IPTG) was purified again on akta and fractions were concentrated and run on SDS PAGE gel
- Conjugation between PAM-Snooptag and beta prefoldin- SnoopCatcher was tested yielding negative results.
- Conjugation between alpha PFD and beta PFD on Native PAGE gel shows good results but need adjustments of protein ratios.
Week 25
30 September - 6 October
Cloning
- Submitted Duke's DBAT, mCerulean3, ATF1 and TycA for sequencing (Ramaciotti Centre).
- Restriction enzyme digest of plasmid DNA. Visualised by gel electrophoresis.
- Sequence alignment check.
- Colony PCR of TycA. Amplicon visualised by gel electrophoresis.
- Preparation of 1000X Ampicillin stocks.
- Small scale grow up of PAM, TycA and gamma-prefoldin DNA. Plasmid DNA extracted and purified by miniprep (QIAGEN). DNA concentration and purity quanitified by nanodrop.
- Transformation of PAM into Origami 2(DE3)pLysS Competent Cells.
Protein Expression & Purification
- Purification of ATF1 for biobrick characterisation was performed.
- Assays on ATF1 activity were unsucessful.
- TycA, DUKE, ATF1 and mCerulean colonies were grown up, expressed and purified with DUKE, ATF1 and mCerulean showing expressed protein determined by bug buster.
- Grow up of alpha PFD beta PFD, PAM, DBAT and ATF1 was performed to keep pellets for later use.
- Beta and alpha PFD was purified on AKTA start machine.
- BCA assay on alpha PFD, beta PFD, mCerulean3 and mVenus gave approximate concentrations of proteins.
- Grow up of alpha PFD beta PFD, PAM, DBAT and ATF1 was performed to keep pellets for later use.
- Native Page was performed on alpha and beta PFD, yielding good results.
- Conjugation between alpha PFD and venus and beta PFD and cerulean yielded good conjugation.
- Concentration and buffer exchange of +c, b+b, mCerulean and Venus.
- Grew up alpha and beta and purified beta. .
- BCA assay was performed on a+c, b+b, mCerulean and Venus to determine protein concentration.
- Ellman assay of ATF1 was performed however was unsucessful
- SDS-PAGE gel of mCerulean3-SnoopTag with BPFD-SnoopCatcher and mCerulean3 (no tag) with BPFD-SnoopCatcher was run to show catcher-tag conjugation however one sample was incomplete.
Week 26
7 October - 13 October
Protein Expression & Purification
- FRET was attempted twice however was unsuccessful.
- Native Page was performed on new alpha and beta PFD protein stocks yielding good results.
- Ellman assay of DBAT was performed however was unsucessful.
- Large scale grow up, espression and purification of PAM was performed.
- SDS-PAGE gel of mCerulean3-SnoopTag with BPFD-SnoopCatcher and mCerulean3 (no tag) with BPFD-SnoopCatcher was run to show catcher-tag conjugation yielding good results.
- Bug buster of DUKE, DBAT and PAM was performed.
- ATF1 was expressed, purified, concentrated, buffer exchnaged and run on BCA assay to determine protein concentration.
- Grow up and expression of PAM in Origami strain was performed twice however appeared to yeild no protein determined by bug buster.
- NMR spectra of alpha and beta-phenylalanine were taken as proof of concept for using NMR for PAM enzymatic assay.
Week 27
14 October - 20 October
Protein Expression & Purification
- Expresseion and purification of DBAT using 6M urea on gravity column.
- Conjugation of DBAT-SnoopTag with BPFD-SnoopCatcher and PAM-SpyTag with aPFD-SpyCatcher using different concentration of Urea was unsucessful.
- SDS-PAGE gel of mCerulean3-SnoopTag with BPFD-SnoopCatcher and mCerulean3 (no tag) with BPFD-SnoopCatcher was run to show catcher-tag conjugation.
- Expression, purification, buffer exchange and concentration of ATF1 was performed.
- Expression and purification, of DBAT was performed.
- NMR spectra of deacetyl-paclitaxel was taken as proof of concept for using NMR for DBAT enzymatic assay.