Notebook

Here's everything we've done on our project day by day.

28/06/2023
  1. PDB pymol images were examined for a template to run through the neural networks pMPNN and RFDiffusion
  2. 6J05.pdb was decided on due to it having all the binding site on the one monomer rather than spread between two dimers
29/06/2023
  1. Interface residues were identified between the monomers of the ArsR template 6J05.pdb using the pymol command
    1. InterfaceResidues 6J05, chain A, chain B)
  2. Interface residues were specified using the pymol script
    1. iterate (chain A and interface and name CA), print(resi)
  3. This produced the list of important interfacial residues between the monomers that would need to be held fixed before being run through the neural network in order to increase the likelihood of dimerisation and therefore binding stability
  4. 7, 10, 11, 14, 15, 17, 18, 19, 20, 23, 26, 27, 30, 81, 84, 85, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 101, 102, 103, 104, 105
  5. The ligand binding site residues were indentified as amino acids 5 angstroms or less from the Arsenic
  6. These residues were as follows; 95, 96, 100, 101, 102, 104.
  7. Therefore total number of fixed residues for the protein before being run through protein MPNN were as follows;
    1. 7, 10, 11, 14, 15, 17, 18, 19, 20, 23, 26, 27, 30, 33, 81, 84, 85, 87, 88, 90, 91, 92, 93, 94, 95, 96, 97, 100, 101, 102, 103, 104, 105
  8. ProteinMPNN model
  9. The 6J05.pdb was run through the protein MPNN online hugging face interface with all previously held residues fixed in order to produce 10 output amino acid sequences
30/06/2023
De novo protein design method:
  1. New RFDiffusion interface was used for de novo protein design link below
    1. Protein Generation via Diffusion in Sequence Space
    2. Input: 6J05 length (105 amino acids long)
        3. Active site residues were indentified in the previous day
      1. 95C, 96C, 100R, 101D, 102C, 104L
      2. In between residues to make one continuous range since, the interface didn't accept discontinuous binding site fragments
      3. 95C, 96C, 97H, 98G, 99T, 100R, 101D, 102C, 103A, 104L
      4. Without amino acids: aa95-104
  2. Four versions of the RFDiffusion models were made, each with slightly different conditions as specified below.
      3. Version (1)
    1. Therefore, to make similar to WT 6J05 94 amino acids before the motif and 1 amino acid after the motif. (3 iterations)
        2. “94,A95-104,1”
      Version (2)
    2. Including the amino acid at 105 from WT structure (3 iterations)
        3. “94,A95-105”
      Version (3)
    3. With a balance of amino acids either side (3 iterations)
        4. “48,A95-104,48
      Version (4)
    4. With a balance of amino acids either side (motif 95-105) (3 iterations)
        5. “48,A95-105,48”
  3. Version 1 and 3 contained the binding motif as specified by the pymol commands
  4. Version 2 and 4 contained one extra amino acid as part of the fixed binding motif - given the binding motif was at the end of the protein, it might be interesting to specifiy the entire tail binding motif.
  5. Version 1 and 2 put the binding motif at the end of the protein as is found in the WT ArsR template.
  6. Version 3 and 4 put the binding motif in the centre of the protein to assess if there was any difference in binding ability
  7. 3 iterations were made for each version and the highest ranking pLDDT was chosen to be ordered
15/08/2023
  1. Inocculation of LB broth with ampicillin and cells (BL21(DE3)+pET21b)
  2. Dilution of gBlocks (gene fragments) to 25 ng/uL in water
16/08/2023
  1. Miniprep of (BL21)(DE3)+pET21b plasmids as backbone for fragment cloning
  2. Restriction enzyme NdeI and XhoI NDE1 reacted with pET21b plasmid to prepare plasmid for cloning
  3. Cultured phi29 (Host: 5547, Bacillus subtilis phage)
22/08/2023
  1. Purification of pet21b backbone by gel electrophoresis
    1. Expected 2 bands, observed 3+ (1.6 Kb, 5.4 Kb, 7 Kb)
    2. Band at 5.4 Kb isolated
  2. Purification of pet21b backbone by centrifugation (Wizard SV Gel and PCR Clean-up System)
23/08/2023
  1. Infused insert with backbone and enzyme and incubated for 15 mins at 50 degrees
  2. Transformed cloned insert fragment into competent stella cells and plated out onto ampicillin agar. Transformations were named as follows (and these are the number codes we use throughout the rest of our project):
  1. ArsR E. coli MX
  2. AfArsR MPNN1 MX
  3. MPNN2 MX
  4. MPNN3 MX
  5. MPNN4 MX
  6. MPNN5 MX
  7. MPNN6 MX
  8. MPNN7 MX
  9. MPNN8 MX
  10. MPNN9 MX
  11. MPNN10 MX
  12. RfDiffusion 4.1 MX
  13. RfDiffusion 2.1 QT
  1. RfDiffusion 2.1 MX
  2. ArsR C. glutamicum QT
  3. ArsR A. ferrooxidans QT
  4. ArsR E. coli QT
  5. AfArsR MPNN1 QT
  6. MPNN2 QT
  7. MPNN3 QT
  8. MPNN4 QT
  9. MPNN5 QT
  10. MPNN6 QT
  11. MPNN7 QT
  12. MPNN8 QT
  13. MPNN9 QT
  1. MPNN10 QT
  2. RFDiffusion 4.1 QT
  3. ArsR C. glutamicum MX
  4. ArsR A. ferooxidans MX
  5. QTEncapsulin
  6. Type 1 EncA
  7. RfDiffusion 3.3 MX
  8. RfDiffusion 3.3 QT
  9. RFDiffusion 1.2
  10. Metallothionein F MX
  11. MT2 MX
  12. MT2 QT (potentially MT1 QT)
  1. Harvested phi29 (Host: 5547, Bacillus subtilis phage)
    1. Phage lysate stored in 4 degree fridge
25/08/2023
  1. Titrated phi29 cell lysate (Host: 5547, Bacillus subtilis phage)
  2. Cultured vB_bSup_Goe1 (Host: 402, Bacillus subtilis phage)
28/08/2023
  1. Innoculating overnight cultures for transformants 1-18: Pick colony from each plate and innoculate 5 mL LBAmp
29/08/2023
  1. Innoculating overnight cultures for transformants 19-34: Pick colony from each plate and innoculate 5 mL LBAmp
  2. Miniprep transformants 1-18: Follow miniprep protocol
30/08/2023
  1. Miniprep transformants 19-35: Follow miniprep protocol
  2. Minipreped sequences for all variants sent off for sequencing
  3. Re-cultured vB_bSup_Goe1 (Host: 402, B. subtilis phage): used 200 uL of phage lysate supplied instead of 100 uL (observed poor phage growth from first culture)
  4. Counted phi29 titration experiment: Current phi29 lysate solution has phage at 4 x 106 PFU/mL (4 degree fridge)
01/09/2023
  1. Harvested phage vB_bSup_Goe1 (Host: 402, B. subtilis phage): stored in 4 degree fridge
  2. Titrated vB_bSup_Goe1 phage lysate: used 200 uL (instead of 100 uL) of 402 B. subtilis for plate culture
04/09/2023
  1. Analysed sequencing: chromatograms really messy, some have the target gene but still not good enough to use. We think that the digestion is where it went wrong so must redo.
  2. Dilute cas9 forward and reverse primers to 1ng/100uL
  3. Inocculation of LB broth with ampicillin and cells (BL21(DE3)+pET21b)
05/09/2023
  1. Miniprep of (BL21)(DE3)+pET21b plasmids as backbone for fragment cloning ("mini pet21" eppendorfs)
  2. Digestion: Restriction enzyme NdeI and XhoI NDE1 reacted with pET21b plasmid to prepare plasmid for cloning
    1. backbone digested for an extra hour on the PCR machine
07/09/2023
  1. Infusion cloning: Very low digested vector concentration (~3ng/µl), so used 7µl plasmid, 1µl insert, 2µl mastermix.
  2. Transformation: Heat shock into stellar E. coli cells. Incubation at 37 °C overnight.
11/09/2023
  1. Inoculations of 1-4, 17-20, 31-34 into 5 mL LB Amp, O/N incubation at 37 °C.
  2. Very low transformation rate of 33. ~10 colonies compared to 100s for other transformants.
13/09/2023
  1. Inoculation of LB broth containing kanamycin with E. coli (containing pRH030 plasmid).
  2. Miniprep of pRH030 plasmids fpr use as backbone for phage gene inserts
19/09/2023
  1. Annealing guide RNA at 95°C for 5 hours and ramping down to 4 °C
  2. Gel purification of digested CRISPR backbone
22/09/2023
  1. Second attempt at ligating gRNAs with CRISPR/Cas9 backbone
    1. Ratio of plasmid:guide:enzyme in ligation = 3:5:2
  2. Transformed ligated DNA into E. coli
26/09/2023
  1. Growing protein cultures (31.2, 20.1, 17.2)
    1. Optimise IPTG induction with 2 diff temperatures (20 °C, 37 °C) and 3 diff IPTG concentrations (0 mM, 0.5 mM, 1.0 mM)
    2. Find best OD and use it's conditions
27/09/2023
  1. Expressed small scale cultures were run on an SDS-PAGE gel to assess which expression conditions was best
    1. The gel didn't show any convincing expression bands - however large scale cultures went ahead due to the time constraints before the final showcase
  2. Glycerol stocks were made for all the recloned samples refer to transformation plate ID:
  3. 400 mL large scale cultures were run for 17.1 20.1 and 31 and induced at OD~0.6 with 1mM of IPTG after ~3 hours of incubation
  4. Large scale cultures were left to express overnight for purification with the AKTA
28/09/2023
  1. Proteins 17.1, 20.1, and 31.2 were purified with the AKTA
  2. SDS page of proteins 17.1 and 20.1 showed no protein expression
29/09/2023

We presented our work at the Australasian Synthetic Biology Challenge final showcase alonside 6 other Australian synthetic biology teams.

03/10/2023
  1. Innoculations of 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2 from glycerol stocks into 5 mL LB Amp
  2. SDS PAGE of 20 (QT Enc) at different stages of NiNTA purification
  3. Heat shock transformation of 17.1, 20.2, 31.1 into Shuffle T7 Express cells, culture overnight on LB Amp plates
  4. Agar gel seperation of digested pET21-GST vector
04/10/2023
  1. Native page is running and should be done tomorrow morning (gel; ladder, A11, A12, B1)
  2. Metallothioneins (36,37,38) and other ArsR variants (15,16,29,30) were cloned and transformed into cells which were plated out - colonies will need to be picked tomorrow and LB cultures inoculated
  3. Shuffle cells were picked and inoculated into LB Kan 6 mL and grown overnight
  4. Miniprepped gRNA plasmids from previous round of transformation (round 2)
05/10/2023
Things to do
  1. Stain Native PAGE
  2. Metallothioneins (LBK) and AsrR (LBA) variants picked and inoculated into LB culture overnight
  3. Shuffle cells (17.1, 20.2, 31.1) and Glycerol stock (18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) primary cultures transfered (100 μL) to secondary cultures (5 mL) and monitored for OD=0.6 for induction by IPTG
Completed
  1. Native PAGE stained - no bands at top of gel unfortunately
  2. Shuffle cells (17.1, 20.2, 31.1) and glycerol stocks (18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) were transfered to secondary cultures and induced at OD ~0.4-0.6
  3. Metallothioneins (LBK) (36, 37, 38) and AsrR (LBA) (15, 16, 29, 30) were picked and inoculated as triplicates overnight (labelled "LBA/K Infusion 36.2"...etc)
  4. Transformed and plated of Stellar E. coli with gRNA Cas9 plasmids (round 3)
    1. Used 2x concentrated gRNA, pRH030 plasmid, ligase, water (diluent) in ratio of 3:3:2:2
06/10/2023
Things to do
  1. Lyse and miniprep Metallothioneins and ArsR triplicates for sanger sequencing
  2. Lyse and denature shuffle cells (17.1, 20.2, 31.1) and Glycerol stock (18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) for a run on an SDS-PAGE gel to assess protein expression
  3. Miniprep successfully transformed Stellar cells with gRNA Cas9 plasmids
Completed
  1. Submitted miniprepped gRNA plasmids (501, 512, 785 - round 2) for Sanger sequencing
  2. Shuffle cells and glycerol stocks (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) run on SDS gels and left to destain over weekend
10/10/2023
Things to do
  1. Check destained SDS gels of (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2)
  2. Try a Western Blot of (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2), it is likely the protein is not overexpressed enough to see results on the SDS gel
  3. Inoculate 5mL primary cultures of all the miniprepped DNA after transformation into Stella cells
  4. Set up Native-PAGE
  5. Maybe make glycerol stocks for Stella cells (17.1, 20.2, 31.1)
Completed
  1. photographed destained SDS gels of (17.1, 20.2, 31.1, 18.3, 1.2, 19.3, 4.2, 2.3, 32.1, 3.2) from Friday
  2. Inocculate 5ml cultures of gRNA Stellar cells (500, 502, 513, 515 x 2, %1 x 3, 785 x 3, 147 x 3 ...) with Kanamycin
  3. Native PAGE of Encapsulins
    1. Ladder - 17.1 (shuffle)- 31.1 (shuffle) - 32.1 (BL21) --- 17.1 - 31.1 - 32.1.
    2. First lot 15 microlitre, second 5 microlitre.
    3. Samples sonicated, then centrifuged. Samples did get warm from sonication.
  4. Transformations of Metallothionein +Enc, metallothionein, some arsR, some de novo into shuffle T7/shuffle T7 express cells
    1. Shuffle T7 Express cells: 31.1 + 38.2, 32.2 + 36.2, 32.2 + 37.1, 36.2, 37.1 (combinations transformed with ampicillin and kanamycin, singles transformed with just kanamycin)
    2. Shuffle T7 cells: 13.2, 15.3, 16.1, 19.1, 20.2 (all transformed with ampicillin)
11/10/2023
Things to do
  1. Stain Native-PAGE
  2. Inoculate Primary cultures into 5mL cultures the morning of all successfully transformed cells
  3. 20.2 and 36.2 failed (will re-transform with 38.2 later today)
  4. Maybe make glycerol stocks for stella cells (17.1, 20.2, 31.1)
Completed
  1. Primary cultures were made for the cells that successfully transformed (13.2, 15.3, 16.1, 19.1, 37.1)
  2. Native-PAGE is being destained
  3. Re-transformed 36.2, 38.2, 29.3, 30.3 and co-transformed 31.1 + 38.2 and 32.2 + 37.1
12/10/2023
Things to do
  1. Inoculate primary 5 mL cultures of re-transformed/ co-transformed plates (36.2, 38.2, 29.3, 30.3, 31.1 + 38.2 and 32.2 + 37.1)
  2. Photograph destained Native-PAGE