Preliminary Research into HIS1 (4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor sensitive enzyme)
Worked on understanding the mechanism of the enzyme
Analysed the structure
Started modelling and docking PP on HIS1
Week 2 (23/06-29/06)
Supervisor meeting
Acquired HIS1 and empty vector (EV) constructs
Expressed the HIS1 and EV constructs
Week 3 (30/06-06/07)
First round of purification. Elution did not show an absorbance peak
More expression of HIS1 and EV constructs and stored cell pellets at -20ᵒC for future use.
Purified the constructs and ran an SDS gel to confirm expression of HIS1
Ran gels for analysis of lysis strategies (sonication vs BugBuster)
Degradation mixtures set up with HIS1 lysate, Empty Vector lysate, and Purified HIS1 with polypropylene film and fibres.
Week 4 (07/07-11/07)
Reaction mixtures were exchanged every day in preparation for GC-MS
Surface Contact Angle (SCA) was optimised with the camera and the light source. Analysis was done with ImageJ DropShape Plugin. Mixtures were exchanged and measurements taken every 24 hours
AFM. Reaction mixtures were set up on 5mg red PP, exchanged after 24h and left over the weekend.
Activity of HIS1 using mass spec was explored, targeting reported substrates like HPP
Preliminary testing of Ni-PV assay. PP fibres were treated with HIS1 lysate and EV lysate. No result as an incorrect blank was used and the spec was unknown as a calibration curve was not done prior.
Week 5 (14/07-18/07)
EV lysate and pure HIS1-treated red PP sent for AFM analysis
Tested lysis methods (sonication and BugBuster) for precipitation of HIS1. Quantification on SDS gels confirms no precipitation
SCA optimisation with camera and light source was continued. At the end of the week, we decided to discontinue this method. The tensiometer was sourced and read for SCA analysis in the coming week.
Ni-PV assay
A calibration curve was made to decipher the appropriate concentration of Ni2+ to use
Deciphered the appropriate volume of reaction mix (with PP fibre) that should be used for the assay.
Another round of testing showed that lysate and FeSO4 were interfering with the assay. So plastics need to be cleaned before testing.
PV dye is light sensitive, so a change in absorbance was measured over 8 minutes. There was no significant change in absorbance.
Set up reaction mixtures on clear PP film
Week 6 (21/07-25/07)
Ni-PV performed on reaction mixtures set up prior. Lysate was removed, and the plastic was cleaned
Optimisation of TLC was performed, using butanol:water: acetic acid for HPP and HGA.
Bake sale for additional funding and networking.
Tensiometer training
Preliminary testing of the Dansyl chloride assay, and tested dansyl chloride with a range of positive controls
Reaction mixture set up with heat-treated PP (200ᵒC for 15 mins)
Used UV-treated samples (PP fibres and PP film) for dansyl chloride assay testing
A single round of IMAC purification of HIS1 to assess activity using the Succinate-Glo assay.
Ni-PV assay with heat-treated PP before and after 24h of incubation in the reaction mixture.
Started cloning vector backbone for downstream cloning -> miraprep protocol did not work, GelRed agarose gels produced streaky bands. Switched to miniprep kit.
Primers arrived, started single-site directed mutagenesis using Bsa1 incorporated primers
Week 7 (28/07 – 01/08)
Reaction mixture with HPP set up for Succinate-Glo assay. Succinate-Glo assay was performed. The calibration curve and samples tested were not successful. Next time, use a white 96-well plate rather than a black one.
Succinate-Glo performed with a white 96-well plate. Again, an unsuccessful calibration curve.
Continued work on the dansyl chloride assay. Testing for a positive control with lactic acid and ethanol.
No success with our positive controls (e.g. Heat treatment and UV treatment). We started planning to use acidified potassium permanganate to oxidise the surface of our PP.
SCA measurements taken of untreated PP using a tensiometer
SCA started on untreated red PP and PP film
SCA was paused to re-evaluate the cleaning and drying process being used due to inconsistent results
Reaction mixtures set up for further analysis
01/08 – AFM results received. Day to decide whether we keep working on HIS1. We decided to continue this work due to the positive results seen from AFM.
Research day on new possible assays.
Miniprep worked; gel purification of the product did not. Re-did miniprep, ran digestion (60, 90, 120 min) with EcoRI and HindIII. Gels showed that 60-minute digestion produced partial digests, but 90 and 120 minutes did not.
Continued working with single-site directed mutagenesis (optimisation with DMSO and Q5 GC Enhancer concentrations). Ordered primers for megaprimer protocol.
Week 8 (04/08-08/08)
04/08-06/08 – Lab closed
Regroup and troubleshoot for assay creation
Purification of EV and HIS1 for Succinate-Glo assay
SCA - Restarted with a new drying and cleaning protocol. Untreated plastic was measured with a new protocol - sonication, oven dry, triplicate within plastic, 7ul drops, bugbuster samples. Pre-KMnO4 plastics were also measured, but only three plastics were measured by mistake; these correspond to the ones that were later treated for 8h by KMnO4, not 4h.
Single-site directed mutagenesis protocol did not work=> switched to megaprimers. Started working on optimizing asymmetric PCR for residue 110 (cycles, anneal temp., etc.)
08/08 – supervisor meeting
Week 9 (11/08-15/08)
Reaction mixtures were set up for the Succinate-Glo assay. Succinate-Glo Assay performed. Calibration curve solutions had a much lower concentration of 2OG (we assume this is what is interfering with results; the 6mM is too much background noise). The calibration curve worked, and no difference was noted between pure HIS1 and EV wells.
More expression of HIS1 and EV
A 2nd round of AFM samples was handed off to Dr Laura Charlton for analysis using AFM
SCA: reaction mixtures were set up, now submerging the plastic. Measurements were taken after 24h, 48h, and 72h of treatment with the new protocol.
FTIR meeting with Dr Simone Dimartino to discuss compatibility with the plastic films.
TLC – optimised now using ethyl acetate:hexane: acetic acid (70:30:2 worked the best).
Continued optimisation of asymmetric PCR protocol => changing extension time from 30 sec to 60 sec, end primer to forward primer ratio of 20:1 to 100:1 resulted in a clear asymmetric band.
Week 10 (18/08-22/08)
Degradations set up for TBO and Ni-PV assays.
Preparations for acidified KMnO4 treatment. We now have positive controls to test our assays. Treated PP samples.
SCA degradations set up. Surface contact final HIS1 and EV measurements taken today after 96h of degradation. Also did KMnO4 4h and 8h treated samples were measured (note: the machine was slightly weird on the day, fit error was higher than usual; maybe discount the 4h ones as we don’t have pre-treatment measurements).
TLCs of HIS1 and EV lysate +/- HPP carried out in triplicate
Received GC-MS results
Second round of AFM discussion with Dr Laura Charlton. Takeaway: There was still residue on our samples interfering with the analysis. Dr Laura Charlton offered to use her cleaning protocol and re-analyse the samples.
Recharging of the IMAC column for next week's purification of HIS1 to set up degradation for FTIR and GC-MS analysis.
TBO - both PP hole punch and PP eppendorf were tested for each sample. Samples = untreated, HIS1 lysate, EV lysate, KMnO4 treated (note: we stopped the HIS1 and EV treated samples mid protocol as we realised we hadn’t cleaned properly due to unusual streaking and the results would not be valid).
Asymmetric product concentration too low for full-length gene protocol => pooled 6 50μL reactions
Initiated full-length gene synthesis for residue 110
Started optimising asymmetric PCR reactions for other target residues
Week 11 (25/08-29/08)
Fresh HIS1 lysate was made up → Purification of HIS1 (double the volume); mistakes were made during the purification of the first set of enzyme; the second set of enzyme was not spun down. The next day, purification was finished with SpinDown.
Gel ran on the lysate to check proper lysis. Lysate degradation solution set up for TBO with fresh HIS1 lysate and EV lysate. Degradation solutions are also set up for FTIR and TBO with fresh pure HIS1. These were exchanged at 24h and 48h. FTIR plastics were ultrasonically cleaned with 1% SDS, distilled water, and ethanol before briefly washing with water again, then drying in an oven at 50 degrees Celsius.
18 FTIR samples were done with (KMnO4, HEPES, Untreated, EV lysate, HIS1 lysate, Pure HIS1)
Full-length gene synthesis worked, but faint bands=> Dpn1, EcoR1, HindIII digest then gel purification
Ligation into backbone, transformation into competent cells=> did not work, due to low backbone concentration => Increasing backbone conc. worked and produced colonies
Moved labs.
Week 12 (01/09-05/09)
Created asymmetric PCR product for residues 110, 148, 268, 297, 304, 305 => tried full-length gene synthesis => failed, no bands observed.
Combinatorial library synthesis deferred due to lack of time.
Sequencing results came back for heterogeneous liquid culture => did not work, sent in samples from individual colonies.
Report writing
Week 13 (08/09-12/09)
Sequencing results for individual colonies worked, but no mutation was observed
All work on directed evolution halted due to a lack of time and further screening methods.
Planning KMnO4 time treatment
KMnO4 timed treatment. (2hrs, 4hrs, 6 hrs, 8hrs).
Report writing
Week 14 (15/09-19/09)
Final AFM results meeting with Dr. Laura Charlton
Final Assay Development Tests (TBO and Nickel-Pyrocathecol Violet) with the timed KMnO4 treatments.