A small-molecule chemical interface for molecular programs


Supplementary Note 1. On switch optimization for working at 37 o C. 
Most PEN DNA based molecular networks reported so far work around 40 o C to 50 o C. Here our goal is to decrease the temperature to 37 o C, in order to bring general compatibility with most known transcription factors which come from mesophilic organisms. Initially, we observed that simply using a reported bistable switch (1) (see Supplementary Table 1), originally designed to work at 50 o C (switch50), led to loss of function when the temperature was lowered. This is shown in Supp. Fig. 1 where the switch50 assemblies containing (on not) the cognate source template is trialled over a range of temperatures. The ability of the switch to start thanks to sTα appears to decay quickly as temperature decreases. At the same time, for temperature of around 42 o C and below, a non-specific start of the circuit (i.e. start in the absence of sTα) appears and becomes dominant. We concluded that at 37 o C the specific and non-specific start times would be bound to overlap. Supplementary Figure 1. Switch50 performance of a range of temperatures. The switch was tested with (circles) or without (squares) source template at various temperatures. The expected behaviour (fast switching in presence of sT and no switching in the absence of sT) is observed down to 42 o C, although the discriminative power seems to deteriorate. Below 42 o C, no discrimination was observed for Switch50. 

Method

The reaction buffer used was identical to the one reported in the main text. However, the enzyme composition was different. Vent (exo-), the nicking endonucleases Nb.BsmI and Nt.BstNBI (all from NEB) were respectively used at 80, 300 and 100 U ml -1 (respectively 4%, 3% and 1% final dilutions of the commercial stock solutions). The thermophilic 5' -> 3' exonuclease ttRecJ was purified in the laboratory, stored in Diluent A (NEB)+ 0.1% Triton X-100 at a concentration of 1.53 µM and used at 22.95 nM as before. aTα was present at 50 nM, pTα at 20 nM, rTα at 50 nM and sTα at 0.5 nM (here used simply as source template). 
In this experiment, Vent exo-seemed less efficient at lower temperatures. Previous PEN-DNA work has been reported with other polymerases such as Bst full length, Bst large fragment (2,3) or Bst2.0 (warm start) ( 4), (all from NEB). Although, Bst Large Fragment is a thermophillic polymerase, we found, in line with others (5), that it had sufficient activity at 37 o C. Klenow was also tested in preliminary experiments, and yielded similar results. Therefore, due to its compatibility with physiological temperature, lack of 3'->5' exonuclease activity and high strand displacement activity, we selected Bst large fragment DNA polymerase for use throughout the study. 
We then designed a new switch using sequences with lower melting temperature, as reported in the main text. We next had to find out the correct balance between aT and pT. Insufficient pT would result in overly weak thresholding and undesired self-starting due to spurious initiation. Excess pT would create an unnecessary drop in sensitivity as stronger stimulation would be required to start the switch. We determined 6 nM of pT to be minimally sufficient (Supp Fig. 2). We chose to proceed with 7 nM of pT to increase robustness of future experiments. Supplementary Figure 2. Fluorescence time-trace of the switch with a range of pT concentrations. The 37 o C reaction was assembled as reported bar only containing aT and rT oligos. Samples with 0, 2 and 4 nM pT start sequentially with little delay whilst 6-14 nM pT samples remain off. The reaction was observed for 15.65 (hr) with no significant further change in fluorescence. 

Oligos used in switch50.


Name Analogues in current work


Supplementary Table 1 .

 1 Oligos used in switch50 as previously reported. Stars represent phosphorothioate modification. Fluorophore and quencher modification positions are shown for rT. Dashes are shown for ease of interpretation. 




  




  




  




  




  




  




  




  




  




  




  


Table of Vectors and genes

 of 
	p070	TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGA CTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAG ACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGA
	Name plasmid	Sequence CCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC GAGGAACTATATCCGGATATCCACAGGACGGGTGTGGTCGCCATGATCGCGTAG AACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTC
	PfB ordered as gBlock provided by Rémi Sieskind, containing sfGFP His-pIVEX vector used in this work	TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA TCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCAAAGCG GTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGG ATGTGGTTCGGTGAGTTTGGTGGGCAGTATGTCCCGGAAACGCTTATTGAACCG GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG GTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATAG AGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAG CTGAAGGAGCTGGAAAAGGCATATAAGCGGTTCAAAGATGATGAAGAGTTCAA ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG CGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCCG GGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATC CCGCCAACTCAACTATTACTTAAAAACATGGGCTGGTCGCCCAACTCCGCTGTATT GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTG CAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGGT AGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGA ATGCTAAGCGTCTTACGGAAAAGATTGGGGGTGCAAAAATCTACCTGAAGCGTG CAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT GACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCTG TGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAAC AAGACTTGGTGCACGGGGGCGCTCATAAAACAAATAACGCGATTGGCCAGGCTT TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT ACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGATA TGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAA TGCTCGCAAAATTTATGGGTAAGACCCGCTTAATTGCTGAGACAGGCGCAGGTC CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT AGCTGTCAAACATGAGAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAATT GGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCA AACATGGCGTAGCCACAGCGATGGCTGGCGCGTTATTAGGCATGAAGGTAGATA TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGC CGACGTTGTAAAACGACGGCCAGTGCCAAGCTTGCATGCAAGGAGATGGCGCCC TCTATATGGGTGCTGAAGACGTAGAGCGCCAGAAGATGAACGTTTTTCGCATGA ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG CTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCC AACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTC AGTTGTTGGGGGCGAATGTAATTCCAGTCAACAGTGGCTCACGCACTTTGAAAG ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC ATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAG ATGCCATTAATGAAGCATTACGGGACTGGGTAGCCACCTTCGAGTACACGCACTA GCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT GCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGC CCTGATTGGCTCTGTCGTCGGCCCTCATCCGTATCCTACTATTGTGCGCGACTTCC ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG GTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGA AGAGCGTCATTGGCCGTGAAGCCAAAGCACAAATCCTGGAGGCAGAGGGCCAG CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC TTGCCTGACGTCATTGTCGCCTGTGTTGGGGGCGGGTCAAACGCTATGGGGATC TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC ATGGAGCTTTTCACTGGCGTTGTTCCCATCCTGGTCGAGCTGGACGGCGACGTAA TTTTACCCGTTTGTTAATGATAAAAAAGTCAAACTCGTTGGCGTGGAAGCTGGCG TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGC GCAAGGGGCTTGAGAGTGGCAAACATTCGGCAAGCTTAAATGCTGGGCAGGTT CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC AAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCA GGGGTATTTCATGGCATGTTATCTTACTTCTTACAGGACGAGGAGGGGCAAATC AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC CCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCA AAGCCAACACACTCGATTGCGCCGGGGCTGGATTACCCTGGTGTCGGGCCGGAA CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGG CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA CACGCTTACCTGAAAAAAATCCAACGTGCGGAGTACGTGACTGTCACAGATGAG GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGA GCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAA GAAGCGCTCAAAGCCTTCCACGAATTATCACGTACCGAAGGGATCATCCCGGCTC CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA CCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC GTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA TGGAGTCAGCACATGCCGTCGCGTACGCTATGAAGTTGGCGAAAGAGATGTCAC CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGA TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTA GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA GTGATGAAATCATTATTGTAAACTTGAGTGGTCGGGGCGATAAAGACCTCGACA TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT GGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGG ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGA TCGTCCTGAAAGTCAGCGGTAATGTG CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC ACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTG TCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA CTGGTGCCGCGTGGCAGCTTACATCCCGGGGGGGGTTCTCATCATCATCATCATC GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTT GTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTG ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCAC ATTAATAAAAGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGGC ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAA CAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT CCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCT TGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATA ACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGA TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGT GGAGTTCGTGACCGCCGCCGGGATCGTACAGACCGGTGAATTCACCATGGACAA ACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAA AACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTC CATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT AGACTGCGAAATGAAGCGCACCACCCTGGATAGCCCTCTGGGCAAGCTGGAACT GGAGGAACTATATCCGGATATCCACAGGACGGGTGTGGTCGCCATGATCGCGTA GTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGG TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTA GTCTGGGTGCGAACAGGGCCTGCACCGTATCATCTTCCTGGGCAAAGGAACATC GTCGATAGTGGCTCCAAGTAGCGAAGCGAGCAGGACTGGGCGGCGGCCAAAGC AGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAG ATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTG TGCCGCCGACGCCGTGGAAGTGCCTGCCCCAGCCGCCGTGCTGGGCGGACCAG GGTCGGACAGTGCTCCGAGAACGGGTGCGCATAGAAATTGCATCAACGCATATA GGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATC ACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT AGCCACTGATGCAGGCCACCGCCTGGCTCAACGCCTACTTTCACCAGCCTGAGGC GCGCTAGCAGCACGCCATAGTGACTGGCGATGCTGTCGGAATGGACGATATCCC AGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGA GCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAA CATCGAGGAGTTCCCTGTGCCAGCCCTGCACCACCCAGTGTTCCAGCAGGAGAG GCAAGAGGCCCGGCAGTACCGGCATAACCAAGCCTATGCCTACAGCATCCAGGG TGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAAC ACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCT CTTTACCCGCCAGGTGCTGTGGAAACTGCTGAAAGTGGTGAAGTTCGGAGAGGT TGACGGTGCCGAGGATGACGATGAGCGCATTGTTAGATTTCATACACGGTGCCT TGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAA CCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAA CATCAGCTACAGCCACCTGGCCGCCCTGGCCGGCAATCCCGCCGCCACCGCCGCC GACTGCGTTAGCAATTTAACTGTGATAAACTACCGCATTAAAGCTTATCGATGAT GGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCA TAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG GTGAAAACCGCCCTGAGCGGAAATCCCGTGCCCATTCTGATCCCCTGCCACCGG AAGCTGTCAAACATGAGAATTCGTAATCATGTCATAGCTGTTTCCTGTGTGAAAT CGACGTTGTAAAACGACGGCCAGTGCCAAGCTTGCATGCAAGGAGATGGCGCCC TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT GTGGTGCAGGGCGACCTGGACGTGGGGGGCTACGAGGGCGGGCTCGCCGTGA TGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG AACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTC CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAG AAGAGTGGCTGCTGGCCCACGAGGGCCACAGACTGGGCAAGCCTGGGCTGGGT CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCC ATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAG AAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCT CCTGCAGGCGGATCCGCGTTTAAACTCGAGGTTAATTAATCCCGGGGGGGGTTC CGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC GCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGC CTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAA TCATCATCATCATCATCATTAATAAAAGGGCGAATTCCAGCACACTGGCGGCCGT GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACT GTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGA GTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA TACTAGTGGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTG CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG CCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGG ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAA TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTC GACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTT GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGC ATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT AAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCC CGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGA TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAA CCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACC CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAA CCACCGCTGAGCAATAA

Table of primers

 of 
	Purpose		Name	Sequence
			Filippo	TGGTATATCTCCTTCTTAAAGTTAAACAAAATTAT
			rev_020	TTCTAGAGGG
	Vector	amplification		CTGGTGCCGCGTGGCAGCTTACATCCCGGGGGG
	(pIVEX)		iR280	GGTTCT
				AAGCTGCCACGCGGCACCAGCACATTACCGCTGA
			PfB2 REV	CTTTCA
				CTTTAAGAAGGAGATATACCATGTGGTTCGGTGA
	Vector integration	PfB2 FWD	GTTTGG
	Point	mutation	T292S_R	TTGCCCCTCCTCGTCCTG
	introduction		T292S_F	ATCAAGCCAAGCCACTCGATTGCGCCGG
			EcB REV2	AAGCTGCCACGCGGCACCAGGATTTCCCCTCGTG
			non-stop	CTT
				CTTTAAGAAGGAGATATACCATGACAACATTACT
	Vector integration	EcB FWD	TAACCCCT
			M149T_F	GTTTTTCGTACCCGCTTAATGGGTGCGGAAG
			M149T_R	GTTAGGCGACTGGCGTTC
	Point	mutation	N171D_F	AGATGCCTGTGATGAGGCGCTGC
	introduction		N171D_R	TTCAGCGTCGCGGAACCG
				CTTTAAGAAGGAGATATACCATGGCCCAACAATC
			GTrp FWD	ACCCTA
				AAGCTGCCACGCGGCACCAGATCGCTTTTCAGCA
	Vector integration	GTrp REV	ACACCT
				CTTTAAGAAGGAGATATACCATGGTGAATGTGAA
			GLacI FWD	ACCAGTAAC
				AAGCTGCCACGCGGCACCAGCTGCCCGCTTTCCA
	Vector integration	GLacI REV	GTC
			iR338	GAAGATGATACGGTGC
	sfGFP amplification control	iR337	TAGAGGATCGAGACCT

Supplementary Information


Table of contents

We started with the design of Source Templates (sT), a hairpin structure, containing an Nt.BstNBI site followed by the B11 signal sequence. Starting from the fully elongated form, sequential nicking, B11 dissociation and polymerase extension result in constant production of B11, proportional to sT concentration. To increase circuit start efficiency, we added 2 bp at the 3'-end, to match aT but not pT. This allowed to bypass the thresholding module and increase affinity for aT. We also shortened B11 sequence on the 5'-end to compensate for 3' extension and ease dissociation from the source template. We propagated the strategy above to Protein Sensing Templates (psTs). The same hairpin structure was taken and TrpR or LacI operator site was inserted between the Nt.BstNBI site and B11 output signal. When operator site is not occupied constant signal production occurs like above. However, this mean that the output sequence is longer as it is now catenated to a part of the operator sequence. Although this makes spontaneous dissociation unlikely (post nick), this issue is alleviated by the fact that Bst polymerase has strand displacing activity. One can observe that the LacI operator site consists of 22 bp and is 3 bp longer than TrpR site. We think this could have contributed towards worse performance of LacI-based templates, by reducing the rate of output production of the corresponding PsT. This constraint was in part mitigated by adjusting concentrations of templates. Protein sensing killer Templates (pskTs) were based on corresponding psT designs with replacement of output from B11 signal to antiB11 (aB11) sequence, similar to the pT sequence. As before, the resulting output contains part of the operator site as a tail. Supplementary Figure 3. In detail representation of the sensing modules used in this work. Here, going from left to right along a hairpin structure: polyT region and 6 bp, providing hairpin backbone; Nt.BstNBI recognition site (underlined); operator domain for TF binding (red), within which Nt.BstNBI nicking site is indicated (red triangle); functional region consisting of pT sequence or modified B11 signal sequence (shown standalone as well). The part of the sequence that was not chemically synthesised, but filled in by DNA polymerase upon reaction initiation, is shown in gray. Dashes are used for ease of interpretation. 1. Small molecule sensing occurs due to unbinding of the transcription factor in response inducer addition. Sequential nicking and extension result in production of B11 "+2". B11 or B11 "+2" can bind aT and become extended. Nicking occurs. Short DNA fragments can dissociate. B11 and B11 "+2" can also bind pT. B11 "+2" is futile. B11 binds pT preferentially and is extended to contain a sequence non-complimentary to aT. B11 or B11 "+2" can also bind rT. B11 "+2" binding is futile. B11 can be extended to permanently open the rT hairpin. All "signal" strands can be degraded by ttRecJ 5' exonuclease whilst "toolbox" strands are protected due to 5' PTO modification. Supplementary Figure 8. Optimizing the sT concentration. We illustrate here the procedure used to determine the concentration of sT that we used throughout the work. For this we carried out a serial dilution of sT, with samples containing or not 5 nM of pskTLacI. We were expecting to see a point where sT concentration would be sufficient to activate the switch, yet would be repressible. We found that a concentration of 8 pM -the minimal attempted -activated the switch with a small delay compared to higher concentrations, for which the triggering effect tended to saturate. In addition, lower concentrations of the sT correlated to better repression by pskT. Therefore, we have chosen the lowest concentration tested. We did not go lower as in potential future emulsion work lower concentrations can be problematic. 

Supplementary Figure 4. Extended version of Figure


Supplementary


Supplementary Note 3. On effect of TrpR concentration on apparent Kd of pskT-based sensor.

In this work, we did not investigate systematically the impact of TF concentration on the circuit response. However, we expect it to affect the apparent Kd. In Supp. Fig. 9, we compare the response to L-trp for two TrpR concentrations, in the case of the inverted design with pskT (positive response). The 2-fold dilution notably affects apparent Kd which increases, as expected, but also the response becomes sharper. A further dilution to 42 nM resulted in loss of discrimination of the circuit as pskT repression became insufficient to overcome inhibition for all L-trp concentrations (not shown). This tuning opens up the possibility to adjust the sensing range and dynamics, depending on the application. Supplementary Note 4. Alternative sensing function shapes to complement Figure 3. 

Supplementary

By adjusting the experimental conditions, it is possible to obtain various shapes for the response of a system to its two inputs. For example, compared to the system shown in Figure 3D, reproduced here as Supp. Fig. 10A, increasing pT concentration from 1nM to 4nM, setting a higher threshold, as well as decreasing psTTrpR from 62.5 pM to 12.5pM, sets a much sharper cutoff in both dimensions (Supp. Fig. 10B). 
In Supp. pskTs tend to provide sharper transitions implementing binary classification (Supp. Fig. 11A). 
The system is very sensitive as doubling sT concentration (from 8 to 16 pM) has a large impact on the classification limits (Supp. Fig. 11B). To obtain the smooth log-linear summing, we used 8 pM sT but rebalanced pskTTrpR, pskTLacI, from 5 nM of each to 7.5 nM of pskTLacI and 2.5 nM of pskTTrpR. This corrects the bias towards stronger TrpR-based element and brings more samples into the window of observation as the total amount of pskT leads to less repression (taking into account that LacI-based elements are less efficient) (Supp. Fig. 11C). Finally, we made a fine adjustment to pskTTrpR=1.5nM, pskTLacI=7.5nM, to further rebalance and speed up the switch to produce the final plot reported. The positive/positive design shown in Figure 3A mixes psT and pskT. Supp. Fig. 12A shows a system starting too fast for input discrimination to occur. Decreasing the amount of psTLacI ten-fold from 2.5 nM slows down the system, but makes it little sensitive to IPTG (Supp. Fig. 12B). Another option is to enter the dynamic range is to increase pT (from 4 nM to 7 nM in Supp. Fig. 12C). This provides a reasonable window of observation, which we narrowed down to arrive to the final plot reported (Supp. 

Supplementary Note 5. Enzyme activity detection and gene amplification response.

In the section we wish to have the output of the PEN-DNA circuit, instead of producing a fluorescent signal, to enable the amplification of a full-length protein-encoding gene. The goal is to obtain an enzymatic activity-to-genetic amplification circuit that can be used to build autonomous selection networks for micro-compartmentalized directed evolution protocols. We thus replace (or complement) the reporter template by two primer-producing templates (ppT), which will use the signal B11 as input, and will produce two primers targeting a given gene. After isothermal incubation, primers are then produced according to the timing of ONswitching of the PEN circuit (with earlier switching leading to more primer accumulation), and can be used during a subsequent PCR. The primer-producing templates are designed as follows: 3' to 5', it contains the B11 binding site, a Nt.BstNBI recognition/nicking site with a 4 base pair spacer, and then the primer encoding sequence (Supp. Fig. 10A). 
Following switch activation, B11 signal binds on ppTs and is extended. Cycle of nicking, product dissociation and polymerase extention will ensue, like previously described for psT. 
The result is a linear production and accumulation of primers, conditional to switch activation (Supp. We tested a pair of ppTs (coding for a primer pair) to couple TrpB activity to gene amplification by PCR. After optimizing the conditions to enable enzymatic activity, isothermal circuit, and PCR in a single buffer, the experiment was conducted one pot, with ppTs at 10 nM and the target gene at 62 pM (see below for detailed reaction conditions). To be able to monitor the reaction in real time, we used both rT and ppT downstream of the circuit. We incubated the MP with a range of EcB concentrations at 37 o C. After full switch activation was observed for the fastest sample, we waited an additional 40 minutes (to enable primer production and switched the machine to PCR-cycling program. The PCR reaction was followed in real time thanks to an intercalating dye. We indeed observed a correlation between switching time and real-time PCR amplification rate (Supp. Fig. 11A-B). We also checked by agarose gel electrophoresis that the PCR reaction produced the expected amplification product (Supp. Fig. 11C). Overall, this proves that we chemically link a catalytic activity, orthogonal to DNA, to gene amplification. 

Method

In this section, a modified assembly was used. The buffer contained 20 mM Tris-HCl pH8.9, 5 mM (NH4)2SO4, 20 mM KCl, 7 mM MgSO4, 5 mM NaCl, 2 μM Netropsin (Sigma-Aldrich), 200 μg ml -1 BSA (New England Biolabs, NEB), 1% Triton X-100 (Sigma-Aldrich), 0.4X EvaGreen dye (Biotium) and dNTPs (200 μM each). As before, the mixture also contained Bst DNA polymerase, large fragment, the nicking endonucleases Nb.BsmI and Nt.BstNBI (all from NEB) respectively used at 20, 200 and 50 U ml -1 (respectively 0.25, 2 and 0.5% final dilutions of the commercial stock solutions). The thermophilic 5' -> 3' exonuclease ttRecJ was purified in the laboratory, stored in Diluent A (NEB) + 0.1% Triton X-100 at a concentration of 1.53 µM and used at 22.95nM. In addition, Vent (exo-) from NEB was used at 20 U ml -1 or 1% of the commercial stock, 50 nM of TrpR dimer, 5 mM of L-serine, 80 μM PLP, 2.5mM indole, 62pM sfGFP-template plasmid (p070 -see Table of Supplementary Note 6. Cloning materials. 
NEB 5-alpha annotated genome (GenBank: CP017100.1) was used for E.coli-sourced parts. 
PfB sequence was inferred from UniProt (Q8U093 (TRPB1_PYRFU)), adjusted by IDT for codon optimisation and synthesis constraints and ordered as gBlock. TrpR, LacI and TrpB coding sequences were cloned into pIVEX vector containing Thrombin cleavage site and His-tag on the C-terminus via Gibson assembly using primers shown below.
Vasily A Shenshin, Camille Lescanne, Guillaume Gines, Yannick Rondelez
A small-molecule chemical interface for molecular programs