Engineering a New Class of Anti-LacI Transcription
Factors with Alternate DNA Recognition
Version 2 2019-01-20, 13:03
Version 1 2019-01-20, 13:03
Posted on 2019-01-20 - 13:03
The
lactose repressor, LacI (I+YQR), is an
archetypal transcription factor that has been a workhorse in many
synthetic genetic networks. LacI represses gene expression (apo ligand)
and is induced upon binding of the ligand isopropyl β-d-1-thiogalactopyranoside (IPTG). Recently, laboratory evolution was
used to confer inverted function in the native LacI topology resulting
in anti-LacI (antilac) function (IAYQR), where
IPTG binding results in gene suppression. Here we engineered 46 antilacs
with alternate DNA binding function (IAADR).
Phenotypically, IAADR transcription factors
are the inverse of wild-type I+YQR function
and possess alternate DNA recognition (ADR). This collection of bespoke
IAADR bind orthogonally to disparate non-natural
operator DNA sequences and suppress gene expression in the presence
of IPTG. This new class of IAADR gene regulators
were designed modularly via the systematic pairing
of nine alternate allosteric regulatory cores with six alternate DNA
binding domains that interact with complementary synthetic operator
DNA sequences. The 46 IAADR identified in this
study are also orthogonal to the naturally occurring operator O1. Finally, a demonstration of full orthogonality was achieved via the construction of synthetic genetic toggle switches
composed of two nonsynonymous unit pair operations that control two
distinct fluorescent outputs. This new class of IAADR transcription factors will facilitate the expansion of
the computational capacity of engineered gene circuits, via the scalable increase in the control over the number of gene outputs
by way of the expansion of the number of unique transcription factors
(or systems of transcription factors) that can simultaneously regulate
one or more promoter(s).
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Rondon, Ronald
E.; Wilson, Corey J. (2019). Engineering a New Class of Anti-LacI Transcription
Factors with Alternate DNA Recognition. ACS Publications. Collection. https://doi.org/10.1021/acssynbio.8b00324
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AUTHORS (2)
RR
Ronald
E. Rondon
CW
Corey J. Wilson
KEYWORDS
operator DNA sequencesADR bind orthogonallyAnti-LacI Transcription Factorsnonsynonymous unit pair operationsDNA binding functionLacI represses gene expressionIPTG binding resultstranscription factorsYQRDNA binding domainsADR gene regulatorsoperator O 1Alternate DNA Recognitionnon-natural operator DNA sequencesligand isopropyl β-ADR transcription factorsarchetypal transcription factor