Incorporation of a Synthetic Amino Acid into dCas9 Improves Control

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Letter

Incorporation of a synthetic amino acid into dCas9 improves control of gene silencing Balwina Koopal, Aleksander J. Kruis, Nico J. Claassens, Franklin Luzia Nobrega, and John Van Der Oost ACS Synth. Biol., Just Accepted Manuscript • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 23, 2019

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ACS Synthetic Biology

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Incorporation of a synthetic amino acid into dCas9 improves control of gene silencing

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Balwina Koopal1*, Aleksander J. Kruis1,2*, Nico J. Claassens1,3, Franklin L.Nobrega1,4 & John van der Oost1

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*- These authors contributed equally to the work.

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ORCID ID:

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Balwina Koopal - 0000-0002-5310-7528

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Aleksander J. Kruis -0000-0002-5279-5488

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Nico J. Claassens -0000-0003-1593-0377

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Franklin L. Nobrega–0000-0002-8238-1083

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John van der Oost - 0000-0001-5024-1871

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Affiliations

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1Laboratory

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Wageningen, The Netherlands

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2Current

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Research, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands

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3Current

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14476 Potsdam-Golm, Germany

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4Current

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University of Technology, Applied Sciences, Delft, The Netherlands

of Microbiology, Wageningen University and Research, Stippeneng 4, 6708 WE,

address:

Bioprocess

Engineering,

Wageningen

University

and

address: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1,

address: Department of Bionanoscience – Kavli Institute of Nanoscience, Delft

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Corresponding author:

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Aleksander J. Kruis- [email protected]

ACS Paragon Plus Environment

ACS Synthetic Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract

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The CRISPR-Cas9 nuclease has been re-purposed as a tool for gene repression

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(CRISPRi).This catalytically dead Cas9 (dCas9) variant inhibits transcription by blocking either

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initiation or elongation by the RNA polymerase complex. Conditional control of dCas9-

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mediated repression has been achieved with inducible promoters that regulate the expression

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of the dcas9 gene. However, as dCas9-mediated gene silencing is very efficient, even slightly

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leaky dcas9 expression leads to significant background levels of repression of the target gene.

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In this study, we report on the development of optimized control of dCas9-mediated silencing

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through additional regulation at the translation level. We have introduced the TAG stop codon

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in the dcas9 gene in order to insert a synthetic amino acid, L-biphenylalanine (BipA), at a

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permissive site in the dCas9 protein. In the absence of BipA, a non-functional, truncated dCas9

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is produced, but when BipA is present, the TAG codon is translated resulting in a functional,

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full-length dCas9 protein. This synthetic, BipA-containing dCas9 variant (dCas9-BipA) could

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still fully repress gene transcription. Comparison of silencing mediated by dCas9 to dCas9-

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BipA revealed a 14-fold reduction in background repression by the latter system. The here

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developed proof-of-principle system thus reduces unwanted background levels of gene

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silencing, allowing for tight and timed control of target gene expression.

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Keywords: CRISPR-Cas; CRISPRi; Synthetic amino acid; Cas9; Gene silencing


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Gene disruption is routinely applied in the characterization of gene function and in metabolic

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engineering strategies. However, the method is irreversible and in the case of essential genes,

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generally lethal1. Gene silencing has been developed as an alternative which does not involve

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the permanent removal of gene function and allows for partial and timed repression of gene

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function. In bacteria, gene silencing has been achieved by the use of riboswitches2, repressible

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promoters, expression of antisense RNAs3 (asRNAs) and antisense transcription4. Recently,

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a catalytically dead version of Cas9 (dCas9) has been used to accomplish the same goal5,6.

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Cas9 is the nuclease that is involved in bacterial adaptive immunity in a CRISPR type II

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system7, which has been repurposed for other applications, notably genome editing8,9,10,11.

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Native Streptococcus pyogenes Cas9 is able to make double-strand DNA breaks in specific

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sequences with great precision, guided by a small CRISPR-RNA (crRNA) that has a

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complementary sequence to the target DNA12. By replacing two catalytic residues, Asp10 and

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His840 by Ala, a dead Cas9 (dCas9) can be generated that lost its cleavage activity but retains

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the ability to specifically bind DNA. When dCas9 is targeted either to the promoter region

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upstream a gene, or to the non-template strand within a gene, it prevents binding of the RNA

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polymerase or transcription elongation, respectively. As such, dCas9 has successfully been

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used to silence gene expression5,6, a technique that is also known as CRISPR interference

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(CRISPRi)13. An advantage of using dCas9 for gene silencing is that any gene may be targeted

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depending on the spacer sequence of the provided crRNA guide14. In contrast, to control gene

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expression using an inducible promoter or a riboswitch, the upstream region of the gene of

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interest needs to be modified, implying that adjusting the targeting specificity for these systems

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is relatively laborious compared to dCas9 targeting. Furthermore, dCas9/guide complexes

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have a very high affinity for a complementary DNA targets15,16 meaning that efficient gene

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silencing can be achieved even with low levels of dCas95. In comparison, asRNAs generally

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need to be expressed in excess compared to the target mRNA17,18,19.

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The high binding efficiency of dCas9 can also be a drawback when used as a gene silencing

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tool. The problem originates from the inherent leakiness of inducible promoters20 that have

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been used to control dCas9 expression21,6. Due to promoter leakiness, there is constant, low

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level dCas9 production in the absence of the inducer, resulting in background gene silencing.

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This problem was partly solved when a single copy of the repression machinery was integrated

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into the E. coli genome21. Peters et al. (2016)22 used leaky chromosomal dCas9 expression in

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B. subtilus in a study of essential genes, which gave about 3-fold repression without induction.

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However, for many experiments the residual gene silencing is undesired as the difference

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before and after induced silencing will be lower and harder to study.



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Methods to control Cas9 activity post-transcriptionally have been established in eukaryotic

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cells23. The major goal of the conditional control has been to reduce off-target effects of

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genome editing24,25. For example, Cas9 variants, containing self-excising inteins (inducing re-

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activation of Cas9), or regulated degradation tags (inducing rapid degradation of Cas9 in the

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absence of a synthetic ligand) have been engineered26,27. Cas9 has also been split into two

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parts that are joined to form a functional protein after stimulation by an external ligand28 or blue

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light29,30. Attempts to use post-transcriptional control of dCas9 activity in E. coli are limited. One

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example is a 4-hydroxytamoxifen-dependent dCas9 that was developed with a high throughput

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screening system in E. coli. While 4-hydroxytamoxifen-dependent gene silencing was

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achieved, there still was some residual gene silencing in the absence of the inducer31. Other

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studies in E. coli corrected for leaky dCas9 expression by introducing mismatches into the

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sgRNA, thereby weakening the interaction with the DNA. As a result, dCas9 became inefficient

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at silencing genes at low protein levels. The latter approach may need to be optimised per

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target gene and sgRNA binding site5. To circumvent this, the red fluorescent protein-encoding

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rfp gene was fused to the 5’-end of an essential gene, and silenced with a tested set of sgRNAs

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with different degrees of mismatches, which enabled tuning of precise and non-leaky gene

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repression32,33. However, the rfp fusions are themselves laborious, and may affect gene

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expression and function. Alternatively, improved control over dCas9 repression in E. coli was

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achieved by expressing asRNA to silence sgRNAs, but this also relies on optimization of the

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designed asRNAs34. In the cyanobacterium Synechcocystis sp. PCC 6083, tighter control over

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dCas9-mediated silencing was achieved by conditional expression of both cas9 and sgRNA

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genes35.

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In this study we improved post-transcriptional control over dCas9-mediated gene silencing in

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E. coli through introduction of a novel regulatory mechanism at the level of translation, thereby

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making dCas9 production dependent on supplementation of a synthetic amino acid, L-

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biphenylalanine (BipA).

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We first established a baseline for further experiments by using (standard) dCas9 controlled

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by an inducible promoter to silence the expression of the mrfp gene. We chose two promoters

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that were reported to give relatively tight control over gene expression: the anhydrotetracyclin

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(aTc)-inducible PLtetO-1 promoter system36,6, and the m-toluate inducible Pm promoter

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system37,38. A CRISPR array containing two spacers that were previously shown to effectively

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silence mrfp expression6 (Supplementary Figure 1) was expressed constitutively. The two

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spacer spacers were not predicted to have any off-target effects39. They also did not contain

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sequences that could lead to a bad-seed effect40. For both promoters we observed substantial

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silencing of mrfp in the absence of the inducer compared to the non-targeting control condition

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without a CRISPR array (Pm promoter: -84% ± 1% of mRFP/OD600; PLtetO-1 promoter: >-99%

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± 0% of mRFP/OD600, Figure 1A; see also Supplementary Figure 2-3A), suggesting that neither

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of the tight promoters is suitable enough for conditional dCas9-mediated gene silencing. We

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aimed to improve this system by adding a translational level of control.

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In the here described system, the synthetic amino acid BipA is incorporated in dCas9 through

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translation of the TAG codon. Commonly, the TAG codon is a stop codon that is recognized

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by release factor 1, which terminates translation41. However, in E. coli C321.ΔA.exp42, all TAG

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stop codons have been replaced by TAA, and release factor 1 has been disrupted. The TAG

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codon was repurposed to code for BipA by providing the appropriate translation machinery: a

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tRNA that recognizes the TAG codon (tRNACUA), and an aminoacyl synthetase (aaRS) that

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charges the tRNACUA with BipA43,44 (Figure 2A). To investigate whether BipA-dependent

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translation of the TAG codon can serve as a better control mechanism over dCas9-mediated

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gene silencing, we substituted the cas9-GAT30 codon, encoding Asp10, with the TAG codon.

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We also included a His840Ala substitution to completely abolish the cleavage activity of Cas9.

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In the presence of the orthogonal translation machinery, a new version of synthetic dCas9 is

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produced, which has BipA incorporated instead of the catalytic Asp10: dCas9-BipA. However,

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this only happens when BipA is supplied to the growth medium (Figure 2A). In the absence of

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BipA, the TAG codon should not be translated and dCas9 should not be produced (Figure 2B).

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Thus, withdrawal of BipA from the growth medium should provide control over dCas9-mediated

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gene silencing, using E. coli C321.ΔA.exp as a chassis.



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Figure 1. Controlling dCas9-mediated mrfp silencing using either inducible promoters or

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conditional translation of the TAG codon. A – E. coli (Pm-dCas9) and E. coli (PLtetO-1-dCas9) grown

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with and without m-toluate or aTc, respectively. B – E. coli (Pspy-dCas9-BipA) grown with and without

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BipA supplementation. Fluorescence and OD600were measured after 7 hours of growth when the cells

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were in the exponential growth phase. Shown are the mRFP/OD600 values, error bars indicate standard

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error of biological duplicates, measured as technical triplicates.

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To test whether dCas9-BipA is still able to silence gene expression, we expressed dCas9-BipA

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under the native, constitutive S. pyogenes promoter5. Figure 1B shows that E. coli(Pspy-

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dCas9-BipA) cells were able to silence mrfp expression almost completely when a CRISPR

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array was present, but only when BipA was supplemented (>-99% ± 0% of mRFP/OD600

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compared to no array; see also Supplementary figure 4A). Since BipA is considerably larger

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and more hydrophobic compared to the negatively charged Asp, it is unlikely that it could

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restore dCas9 cleaving activity. In fact, we did not observe a growth defect in antibiotic-

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containing medium when both BipA and the CRISPR array were present (Supplementary

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Figure 4B), which indicates that dCas9-BipA did not cleave the plasmid encoding the antibiotic

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marker and mrpf. This result shows that translational control of dCas9-BipA may serve as an

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alternative or addition to transcriptional control. However, we still observed some mrfp

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silencing without supplementation of BipA (-35% ± 1% of mRFP/OD600 compared to no array;

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Figure 1B). This can be explained by non-specific activity of the aaRS/tRNACUA-pair43. When

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BipA is not available, the aaRS may charge the tRNACUA with a native amino acid. This is likely

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Tyr, as the aaRS used in this study is derived from an aaRS that charges the tRNACUA with

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Tyr. Both transcriptional and translational control of dCas9-BipA production thus showed a

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certain degree of leakiness.


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To overcome this, we combined BipA-dependent dCas9-BipA production with inducible dcas9-

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bipA expression under the control of either the PLtetO-1 or the Pm promoter. This resulted in

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optimal control over mRFP production. Adding both the inducer and BipA to our system

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resulted in virtually complete mrfp silencing for both the Pm promoter (-97% ± 1% of

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Figure 2. Combined transcriptional and translational control of dCas9-mediated gene silencing.

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The TAG codon is inserted in the dcas9 gene and can be translated to incorporate L-biphenylalanine

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(BipA) when the suitable translation machinery is provided: a tRNACUA that recognizes the UAG codon

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and an aminoacyl-synthetase (aaRS) that charges the tRNA with BipA. To facilitate translation of the

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TAG codon, E. coli C321.ΔA.exp42 is used as a chassis. An inducible promoter is used to drive dCas9

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expression as an additional control mechanism. A – Without gene inducer or BipA, no dCas9 or mRFPis

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produced. B –When both BipA and the inducer are provided, dCas9-BipA is produced, which silences

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mRFP production. C – Constructs used in this study.

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mRFP/OD600 relative to no array) and the PLtetO-1 promoter (-98% ± 0% of mRFP/OD600

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compared to no array) (Figure 3; see also Supplementary Figures 5-6). In the absence of the

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inducer and BipA, we observed little mrfp silencing when the Pm promoter was used (-6% ±

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3% of mRFP/OD600 compared to no array). Changing to the PLtetO-1 promoter also led to an

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improvement of our system, although there was more mrfp silencing in the absence of the

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inducer and BipA (-16 ± 2% of mRFP/OD600 relative to no array) compared to the Pm promoter.



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In fact, thePLtetO-1 promoter did not require induction to fully silence mrfp in the presence of

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BipA (Figure 3). This indicates that the PLtetO-1 promoter is more leaky than the Pm promoter,

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which is in agreement with our previous results (Figure 1A).

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Surprisingly, supplementation with m-toluate, aTc or BipA lowered mRFP production roughly

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10-20% in E. coli strains when no CRISPR array was present (compared to non-supplemented

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controls). When both an inducer and BipA were supplied, the reduction in mRFP was additive.

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This unspecific effect was observed in both dCas9 and dCas9-BipA producing E. coli strains

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(Supplementary Table 1). In the case of m-toluate and aTc, it is possible that the additional

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burden of dCas9 production diverted cellular resources from mRFP production. Furthermore,

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it was shown that high dCas9 production in E. coli causes growth defects and other changes45,

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which is in line with our observations (Supplementary Figures 2B-6B). This might be alleviated

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by lowering dCas9 expression, which should still preserve the strong repression33. However,

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the observed slight mRFP repression by BipA in the regular dCas9 strain cannot be explained

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by increased dCas9 (Supplementary Table 1). BipA did not have negative effect on E. coli

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growth (Supplementary Figures 2B-6B), demonstrating it is unlikely that the reduced mRFP

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production is due to BipA toxicity. Actually, strains producing regular dCas9 cultivated in the

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presence of BipA grew a bit faster for unknown reasons. These small increases in OD600 can

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significantly impact mRFP/OD600 values and may explain the reduced mRFP/OD600 production.


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Figure 3. Combining conditional translation of the TAG codon and an inducible promoter

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provides optimal control over dCas9-mediated mrfp silencing. A - E. coli (Pm-dCas9-BipA) and B

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- E. coli (PLtetO-1-dCas9-BipA) cells were grown in the absence or presence of BipA and/or the inducer.

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Fluorescence and OD600 were measured after 7 hours of growth when cells were in the exponential

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growth phase. Shown are the mRFP/OD600 values, error bars indicate standard error of biological

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duplicates, measured as technical triplicates.

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Combining transcriptional and translational control over dCas9-BipA production improved

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control over the system, with virtually complete repression when induced, and minimal

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leakiness in the absence of both inducer and BipA. However, the experiments have so far

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focused only on situations where all components were present from the beginning. In practice,

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it may be desirable to regulate gene expression in time. We therefore tested whether mRFP

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production could be stopped in actively growing of E. coli(Pm-dCas9-BipA) cultures. BipA and

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m-toluate were supplemented four hours after inoculation. We observed that the mRFP/OD600

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started decreasing approximately one hour after BipA and m-toluate were added. After 13

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hours, the mRFP/OD600 dropped back to its initial value (Figure 4A). Overall, the results of this



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experiment match the observations in Figure 3. mRFP production was completely diminished

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after adding m-toluate and BipA, and partially repressed when only one of the components

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was present. Interestingly, for the negative control mRFP production was stable for a short

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time, shortly after the other samples were induced (to the negative control only solvents were

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added). This period coincides with the exponential phase of growth. The exponential phase

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was also observed to give temporary stable mRFP/OD600 in other experiments with induction

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from the start (Supplementary Figures 2A-6A). We also observed that the cultures were

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growing rather slowly in general. This was probably caused by the cost of maintaining three

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plasmids, overexpressing multiple proteins (including dCas9) as well as poor aeration from

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growing in the microplatereader46. Regardless, our results emphasize that timed control of

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mRFP production could be achieved by supplementing BipA and m-toluate to the system.

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Figure 4. dCas9-BipA allows for temporal control of mRFP production in actively growing cells.

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E. coli(Pm-dCas9-BipA) was cultivated in LB medium. After 4 hours, m-toluate, BipA or their solvents

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were added (black arrow). Fluorescence and OD600were monitored over time. The values represent

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averages of biological duplicates, measured as technical triplicates. All replicates showed the same

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trends. Error bars were omitted to improve figure clarity but are available in Supplementary Figure 7. A

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– mRFP/OD600values observed during 14 hours of cultivation. B – OD600 values observed during 14

239

hours of cultivation.

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In this study, we demonstrated a new system for inducible gene silencing using a novel,

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synthetic form of dCas9, which has the synthetic amino acid BipA incorporated: dCas9-BipA.

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The novel dCas9 variant retains the ability to silence gene expression and is dependent on

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BipA supplementation. Thus, it may serve as an alternative to transcription-controlled dCas9-


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expression. However, the BipA dependency is leaky; without supplementation of BipA to the

245

medium, there is still some gene silencing. When translational control over dCas9 production

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was combined with transcriptional control using an inducible promoter, the leakiness was

247

reduced significantly. We observed that pairing BipA-dependency with the Pm promoter gave

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the best results. It greatly reduced background gene silencing while enabling almost complete

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silencing when the system was fully induced. We used mrfp to demonstrate the usefulness of

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the system, but in principle any gene (in this E. coli strain) can be targeted. This may be

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particularly facilitated by the novel Cas9 versions that have been developed with different PAM

252

preferences47,48,49,50.

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Alternative solutions based on post-transcriptional (d)Cas9 control have focused on reducing

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Cas9 off-target cleavage in eukaryotic cells. Cas9 has been engineered to switch between an

255

inactive and an active state by means of external stimuli, such as small ligands or blue light.

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An advantage of these systems is that the response to the stimulus may be faster since Cas9

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does not need to be synthesised de novo. However, most of the conditional Cas9 variants

258

retain at least some level of cleavage activity in their “inactive” form51,24,26. If these concepts

259

were applied in E. coli for dCas9-mediated gene silencing, the residual activity may cause

260

unwanted repression, as opposed to the system developed in this study. On the other hand,

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there may be opportunities to combine transcriptional and post-transcriptional control as well

262

with these systems. Another demonstrated approach to prevent unwanted repression by

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dCas9 is to control both dcas9 and sgRNA expression by two different inducible promoters35.

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The here described system relies on a genetically recoded strain for efficient translation of the

265

TAG codon, which may be considered a drawback for general applicability. However, other

266

orthogonal aaRS/tRNACUA-pairs, recognizing for example non-natural 4-base-frameshift

267

codons52, could be used to introduce BipA, or other unnatural amino acids, into dCas9. This

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would potentially allow the system to be used in any E. coli strain. Furthermore, our design

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may also find applications in controlling the activity/expression of other Cas9 versions and

270

even other proteins.

271



272

Methods

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Plasmids and strains

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Table 1. Plasmids used in this study. Plasmid

Use (antibiotic)

Page 12 of 20

Source

pdCas9 (Addgene plasmid # 46569) Construction of pET26B-dCas9 Bikard et al. (2013)5 variants (Cam) pET26B

Construction of pET26B-dCas9 Novagen variants (Kan)

pET26B-dCas9-GCT10TAG

mrfp silencing (Kan)

This study

pET26B-dCas9-GCT10TAG-


This study


This study

-dCas9- mrfp silencing (Kan)

This study

RFParray pET26B-XylS-Pm-dCas9 pET26B-XylS-Pm GCT10TAG pET26B-XylS-Pm-dCas9-RFParray


This study

pET26B-XylS-Pm-dCas9-


This study

pET26B- PLtetO-1-dCas9


This study

pET26B-PLtetO-1-dCas9-


This study

pET26B- PLtetO-1-dCas9-RFParray mrfp silencing (Kan)

This study

pET26B-PLtetO-1-dCas9-

This study

GCT10TAG-RFParray

GCT10TAG mrfp silencing (Kan)

GCT10TAG-RFParray pEVOL-BipA

Translation of the TAG codon Mandell et al. (2015)44 (Cam)

pBbS5a-RFP (Addgene plasmid # Reporter for mrfp silencing (Amp) Lee et al. (2011)53 35283) 275 276


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277 278


Table 2. E. coli strains used in this study. Negative controls used lack the CRISPR-array for targeting mrfp. E.coli strain Strain genotype Source C321.ΔA.exp(Addgene

E. coli MG1655 Δ(ybhB-bioAB)::zeoRΔprfA; Lajoie

et

strain #49018)

all 321 TAG codons changed to TAA

XylS-Pm-dCas9

E. coli C321.ΔA.exp carrying: pET26B- XylS- This study

al.

(2013)42

Pm-dCas9-RFParray, pEVOL-BipA, pBbS5a-RFP PLtetO-1-dCas9

E.

coli

C321.ΔA.exp

carrying:

PLtetO-1-dCas9-RFParray,

pET26B- This study

pEVOL-BipA,

pBbS5a-RFP dCas9-BipA

E.

coli

C321.ΔA.exp

carrying:

pET26B- This study

dCas9-GCT10TAG-RFParray, pEVOL-BipA, pBbS5a-RFP XylS-Pm-dCas9-BipA

E. coli C321.ΔA.exp carrying: pET26B-XylS- This study Pm-dCas9-RFParray, pEVOL-BipA, pBbS5aRFP

PLtetO-1-dCas9-BipA

E.

coli

C321.ΔA.exp

carrying:

PLtetO-1-dCas9-RFParray,

pET26B- This study

pEVOL-BipA,

pBbS5a-RFP 279 280

Strain and plasmid construction

281

E. coli C321.ΔA.exp42 and adk.d6_tyrS.d844 were received from Prof. George Church (Harvard

282

University, Boston), and were used to perform gene silencing experiments and to obtain

283

pEVOL-BipA, respectively. PCR reactions were performed using Q5 polymerase (NEB).

284

Fragments were either purified using the Zymo PCR cleanup kit or excised from gel and

285

purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey Nagel). Ligation was

286

performed using T4 ligase (Thermo Fisher) and restriction-digestions were performed using

287

NEB restriction enzymes in their accompanying buffers. Colony PCR was performed using

288

OneTaq polymerase (NEB), followed by Miniprep using the GeneJET Plasmid Miniprep Kit

289

(Thermo Fisher). Candidate clones were verified by Sanger sequencing.

290

pET26B-dCas9 was constructed using restriction/digestion cloning, using parts of

291

pdCas95 and pET26B (Novagen). To make mutations in dCas9, mutagenesis PCR was used

292

based on the Agilent Quickchange protocol. A CRISPR array of two spacers targeting the non-

293

template strand of mrfp6 was inserted in pET26B-dCas9 and pET26B-dCas9-GCT10TAG by

294

annealing two complementary oligonucleotides (Supplementary Table 3) and cloning them



295

using a Golden-Gate-like protocol54. The constitutive dCas9 promoter was replaced by either

296

the Pm37 (obtained from pHH100) or the PLtetO-1 promoter (as was used by Qi et al. (2013)6,

297

ordered as a gBlock from IDT). The vectors were assembled by Gibson assembly using the

298

Hifi DNA assembly cloning kit (NEB). Assembled plasmids were either transformed into

299

commercial chemically competent DH5α cells (NEB) according to the manufacturers protocol.

300

E. coli strain C321.ΔA.exp was transformed according to the protocol described in Lajoie et al.

301

(2013)42.

302 303

Strain cultivation and fluorescence assays

304

E. coli strains were routinely cultured in LB, supplemented with, chloramphenicol (25 ug/mL),

305

kanamycin (50 ug/mL), ampicillin (50 ug/mL) andbleomycin (5 ug/mL), where appropriate.

306

Gene silencing experiments were performed by cultivating strains in 96-well plates (Greiner)

307

in a SynergyMx platereader (BioTek), at 37°C while shaking continuously. OD600 and mRFP

308

fluorescence (excitation: 584nm, emission: 607nm) were measured every 30 min and 2 µL

309

overnight precultures (in LB) were inoculated into 200µL LB, supplemented with IPTG (100

310

µM), BipA (Fluorochem; 200uM), anhydrotetracycline (aTc; 1 µM) or m-toluate (2 mM), where

311

appropriate. Alternatively, BipA and aTc or m-toluate were added after 4 hours of growth in the

312

same concentrations.

313

314

Acknowledgements

315

We would like to thank Prof. G. Church for his kind donation of E. coli C321.ΔA.exp (Addgene

316

strain #49018), E. coli adk.d6_tyrS.d8 and plasmid pEVOL-BipA. We are grateful to Dr. R. Lale

317

for plasmid pHH100 and to Prof. J.D. Keasling for plasmid pBbS5a-RFP. pdCas9 was a gift

318

from Luciano Marraffini (Addgene plasmid #46569). Lastly, we would like the Wageningen

319

iGEM team 2016 for their support.

320

J.v.d.O. is supported by the Netherlands Organization for Scientific Research (NWO) through

321

a TOP grant (714.015.001) and a Gravitation grant (024.003.019).

322 323 324


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325

Supporting information.

326

Additional figures and tables.

327

Supplementary methods.

328 329



330

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Incorporation of a Synthetic Amino Acid into dCas9 Improves Control

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