Lab notebook

May

Lab introduction
Cloning first level 0 (pES0001-pES0022) and level 1 constructs (pES1001-pES1027)
Cloning of the SP plasmids coding for SIAH1 and SIAH2
Set up the first PACE reactor to evolve base editor TadA, based on a previous work by Richter et al.¹

Cloning most level 1 (pES1028-pES1070) and first level 2 constructs (pES2001-pES2011)
Production of phages bearing different SP plasmids coding for SIAH1 and SIAH2
Troubleshooting of the PACE reactor setup and code

Cloning of level 1 (pES1067-pES1103), and level 2 constructs (pES2012-pES2022), among which:
Side-directed mutagenesis of EGLN3 to disrupt the degron motif
Co-transformation of pES1076+pES2008 (containing EGLN3 as a substrate) into S2060 cells
First propagation assay comparing SIAH1-SP, SIAH2-SP, and UN-SP
Set up PACE for evolution of SIAH1 using S2060 cells co-transformed with pES1076+PES2008
Troubleshooting of the PACE reactor setup and code

Cloning of level 1 (pES1104-pES1111) and level 2 (pES2023-pES2037) constructs, among which:
Level 2 constructs with degron-mutated EGLN3 variants
Level 2 construct without N-term RNAP
While SIAH1-SP phages do not wash out from the PACE lagoon, subsequent phage propagation assays show non-specific propagation ability, so the evolution is put on hold
Co-transformation of pES1035+pES2009 (containing α-Synuclein as a substrate) and pES1033+pES2009 (containing EGLN3 as a substrate) into S2060 cells
Co-transformation of degron-mutated EGLN3 variants (pES1076, pES1097, pES1098, pES1101, pES1102) with pES2008 into S2060 cells
Phage propagation assay with EGLN3 and α-Synuclein as substrates
Phage propagation assay with degron-mutated EGLN3 variants
Phage propagation assay for different sizes of the SP
Set up PACE for drift of SIAH1-SP using S2060-DP6 strain
As the PACE reactor needs further troubleshooting, paralelly set up PANCE for drift of SIAH1-SP using S2060-DP6 strain

Cloning of level 0 (pES0023-pES0028), level 1 (pES1112-pES1158) and level 2 (pES2038-pES2074) constructs, among which:
Constructs with NLRP3 as a substrate
Constructs with different promoter potencies controlling expression of E1 and E2
Constructs containing the pVan + VanR inducible system controlling expression of RNAP subunits
Constructs containing the psp promoter controlling expression of RNAP subunits
Level 2 constructs with SmR (spectinomycin resistance cassette)
Level 2 constructs with different linkers for the N-term RNAP subunit - Ubiquitin fusion
Troubleshooting of cloning system using both Golden Gate and Gibson assembly protocols, including:
Adaptation of the termocycler protocol for Golden Gate assembly
Changing the resistance cassette for level 2 constructions to SmR
Production of a knockout SIAH1 SP plasmid (SIAH1-KO) to validate our system
Co-transformation of pES1076+pES2037 (no N-term RNAP system) and transformation of pES2008 only (no C-term RNAP system) in S2060 cells
Phage propagation assay in cells without either the N-term or the C-term RNAP subunit
Phage propagation assay for vanillic acid-inducible system validation
Assay to confirm that vanillic acid and other components of the system (e.g., RNAP subunits) are not toxic for cell growth

PANCE for different S2060 co-transformant strains using SIAH1-SP for system validation

Richter, M.F., Zhao, K.T., Eton, E. et al. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat Biotechnol 38, 883–891 (2020). https://doi.org/10.1038/s41587-020-0453-z ↩