This document describes a phagemid-based directed evolution system for engineering new transcription factors. The system uses a phagemid plasmid that packages the gene for the evolving transcription factor. Selection is based on the factor's ability to upregulate a "missing" phage gene on an accessory plasmid needed for phage replication. This enriches variants with desired activity over multiple rounds. The system was used to generate orthogonal transcription factors and tune their specificity and strength. It can function in batch or continuous mode and with combinatorial libraries and mutagenesis devices to further improve factors for applications like synthetic gene networks.
3. Lambda cI is a tunable activator or repressor
• Functions as transcription activator or repressor
• One of the best studied transcription factors (structural information)
• Already applied in various synthetic gene circuits
• Stronger activator cIopt available (Bushman, 1989)
4. MI3 Phage
Continuous phage
evolution: PACE
Esvelt et al., A system for the continuous directed
evolution of biomolecules. Nature 472, 499-503
(2011).
6. Basic principle for TFphagemid selection
• Start with a phage library which contains variants of your desired target gene
• Target protein upregulates missing phage gene (e.g. Gene VI)
• Link between protein activity and phage production
• This leads to an enrichment of the protein with a desired activity over time
conditional gene for phage
to complete phage life cycle
7. Characterisation of the system (batchculture)
• Gene III versus Gene VI as “missing gene” on Accessory Plasmid
• Successful enrichment of target gene (cIopt/RFP) with Gene VI
9. Combinatorial cI libraries
• Randomise residues that make direct contact with promoter
• DNA library ->M13 phagelibrary
Stayrook et al., Nature 2008 Albright and Matthews, PNAS 1998
(Val47)
10. Building orthogonal synthetic promoters for new TFs
• Build new promoters based on consensus sequence (CS)
Consensus (wild-type):
Synthetic promoter (5C6A):
Confirmed loss of WT cI binding to new
promoters by reporter assay:
11. Selection against syntheticpromoters
• Positive selection of cI library to activate synthetic promoters
• Counterselection against wild-type cI
• Analysis after 6-8rounds of selection (infection, growth, reinfection)
Positive Selection
Counterselection
Gene VI completes the
phage life cycle thus
promoting replication of
functional cI variants
Any residual wt binding
activity represses Gene VI
and reduces phage
production for that variant
14. Continuous selectionsystem
In collab. with Alfonso Jaramillo’s group (University of Warwick)
Sterile air
supply
Waste
(Kan + CA) (~1.5 Vol per h) (e.g. Ara) (~1-2 Vol per h)
HP AP HP AP PM
2xTY medium Chemostat Inducer Cellstat
HP AP PM
37°C 30°C
cI TF
15. Continuous library selection
• Selection against synthetic promoter PM,4G5T
• Continuous selection for 90 hours (MOI ~ 6)
• Selected library members activate PM,4G5T
Position 44 45 46 47 55
4G5T,P
4G5T,P
cIopt Q S G V N
cI 1 P F S V M
cI 2 Q R R K P
18. Directed evolution withMP6
• cI4A5T6T,P: Least active TF in orthogonalset
• Directed evolution against promoter PM,4A5T6T
with cI4A5T6T,P phage population
• Use of mutagenesis plasmid MP6 results in
further improvement of TF activity
cI4A5T6T,P
T42
T W Q N R I C A A
Position 42 43 44 45 46 47 48 49 55
cIopt M G Q S G V G A N
cI4A5T6T,P M W* Q N R I C A A
Randomised in library (underlined)
Not randomised*
New mutation: T
19. cI variants for downstream engineering of gene networks
• 3 inputs and 2 outputs
• Network with eight proteins in a single cell (3 cI variants)
• Inputs controlled by 0.1% Ara, 10µM IPTG, 1µM 3OC6-HSL
20. Summary
• Engineered a set of orthogonal dual activators and repressors
• Based on a new phagemid-based evolution system
• Evolution system functions in batch and continuous mode
• System for combinatorial libraries and a mutagenesis device
• Constructed single-input and multi-input synthetic promoters
• Tested applicability in model gene networks
Brödel, A.K. et al. Nature Communications 7, 13858 (2016).
Brödel, A.K. et al. Nature Protocols 12, 1830-43 (2017).
21. Thanks
• EVOPROG partners (www.evoprog.eu)
• BBSRC (EvoEngine)
• Alfonso Jaramillo (University of Warwick)
• Andreas Broedel (Imperial)
22. Alternative mutagenesisdevices
• Mutagenesis device used to induce mutations in the target gene
• Can be used as stand-alone or in combination with combinatorial libraries
• Different mutagenesis devices available (e.g. MP4, MP6, EPpolA)
23. • Mutagenesis genes under an inducible promoter (pBAD) which functions as on/off switch
• Performance of mutagenesis system tested via reporter assays
• Ampicillin resistance assay (targets plasmid;; Readout: GAA ->TAA in Glu26 ofβ-lactamase)
• Rifampicin resistance assay (targets genome;; 39 single bp mutations in rpoB gene lead to resistance to rifampicin by
altering β subunit of RNA polymerase)
1.E-07
1.E-08
1.E-09
1.E-10
1.E-11
1.E-12
Ratio(Ampicillinresistantcells)
1.E-13
1.E-12
1.E-11
1.E-10
Ratio(Rifampicinresistantcells)
1500x
3800x
9100x
1x
1x 1x
3x
5x
66x
104x 133x
Alternative mutagenesisdevices
24. Phagemid-based selection system
PM Phagemid: codes for evolving protein and gets packaged in phage
AP Accessory Plasmid: geneVI expression to complete phage life cycle only if evolving protein functions
HP Helper Phage: all other phage genes
25. Presented in the Synthetic Biology &
Gene Editing strand of the 4Bio Summit.
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