Synthetic biology tools

Abstract

Methods for design of genetic circuits are provided.

1 . A method of designing a genetic circuit containing one or more orthogonal sequence-specific DNA binding polypeptides, the method comprising
 providing a set of sequence-specific DNA binding polypeptides;   optimising expression of the polypeptides in a heterologous host cell;   identifying target DNA sequences to which the polypeptides bind;   generating synthetic transcriptional regulatory elements comprising at least one identified target DNA sequence, wherein the regulatory elements are responsive to a sequence-specific DNA binding polypeptide from the set of sequence-specific DNA binding polypeptides;   designing cognate sequence-specific DNA binding polypeptide-target DNA sequence pairs to generate a set of orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs;   designing a genetic circuit containing one or more orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs from the set of orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs, thereby designing a genetic circuit containing one or more orthogonal sequence-specific DNA binding polypeptides.   
     
     
         2 . The method of  claim 1 , wherein the sequence-specific DNA binding polypeptides are selected from the group consisting of transcription factors, transcriptional activators, RNA polymerases, and transcriptional repressors. 
     
     
         3 . The method of  claim 2 , wherein transcriptional repressors are substantially identical to the Tetracycline repressor (Tet R ). 
     
     
         4 . The method of  claim 1 , wherein the host cell is a prokaryotic cell. 
     
     
         5 . The method of  claim 1 , wherein the host cell is a eukaryotic cell. 
     
     
         6 . The method of  claim 1 , further comprising testing the circuit for unintended interactions within the circuit and/or between the circuit and the host cell genome. 
     
     
         7 . The method of  claim 1 , wherein the providing comprises algorithm-guided identification of sequence-specific DNA binding polypeptides from one or more sequence database. 
     
     
         8 . The method of  claim 7 , wherein the algorithm identifies amino acid sequence similarity with a known sequence-specific DNA binding polypeptide. 
     
     
         9 . The method of  claim 8 , wherein a phylogenetic tree is used to maximize the diversity between sequence-specific DNA binding polypeptides in a library. 
     
     
         10 . The method of  claim 7 , wherein the algorithm identifies sequence-specific DNA binding polypeptide based on a phylogenetic tree. 
     
     
         11 . The method of  claim 7 , wherein the algorithm identifies sequence-specific DNA binding polypeptide based on their predicted ability to bind to different target DNA sequences. 
     
     
         12 . The method of  claim 11 , where the predicted ability is based on a bioinformatic algorithm that predicts the target DNA sequence by assuming that the sequence-specific DNA binding polypeptide is autoregulated. 
     
     
         13 . The method of  claim 1 , wherein the optimizing comprises codon optimization of a gene encoding the polypeptide. 
     
     
         14 . The method of  claim 1 , wherein the optimizing comprises selecting random codons different from the native codons such that the coding sequence for the sequence-specific DNA binding polypeptide is different from the native coding sequence. 
     
     
         15 . The method of  claim 1 , wherein the optimizing comprises using an algorithm to eliminate transcriptionally functional sequences in a gene encoding the polypeptide. 
     
     
         16 . The method of  claim 15 , wherein the functional sequences are ribosome binding sites, regulatory elements, or terminators. 
     
     
         17 . The method of  claim 15 , wherein the functional sequences are target DNA sequences for other sequence-specific DNA binding polypeptides in the orthogonal set. 
     
     
         18 . The method of  claim 1 , wherein the target DNA sequences are determined by an in vitro method. 
     
     
         19 . The method of  claim 18 , wherein the in vitro method comprises contacting a set of sequence-specific DNA-binding polypeptides to an array of polynucleotides, thereby determining polynucleotide sequences bound by the DNA-binding polypeptides. 
     
     
         20 . The method of  claim 19 , wherein the array of polynucleotides is a microarray. 
     
     
         21 . The method of  claim 19 , wherein the polynucleotides to form a hairpin. 
     
     
         22 . The method of  claim 21 , wherein the hairpin comprises a target DNA sequence. 
     
     
         23 . The method of  claim 21 , wherein the hairpin comprises a 30 bp inverted repeat. 
     
     
         24 . The method of  claim 23 , wherein the inverted sequence has a T at position 14, A at position 13, A at position 7, T at position -7, T at position -13, and A at position -14. 
     
     
         25 . The method of  claim 23 , wherein the hairpin sequences are designed to have no more than a particular GC content. 
     
     
         26 . The method of  claim 25 , where the GC content of the hairpin is equal or less than 35%. 
     
     
         27 . The method of  claim 18 , wherein the in vitro method is based on high-throughput sequencing to quantify RNA transcripts. 
     
     
         28 . The method of  claim 1 , wherein the target DNA sequences are determined by an in vivo method. 
     
     
         29 . The method of  claim 28 , wherein the in vivo method comprises expression of the sequence-specific DNA binding polypeptide. 
     
     
         30 . The method of  claim 28 , wherein the in vivo method comprises constructing a synthetic regulatory element library, wherein regulatory elements in the library comprise one or more of the identified target DNA sequence(s). 
     
     
         31 . The method of  claim 30 , wherein the synthetic regulatory element library comprises mutations in the target DNA sequence binding region. 
     
     
         32 . The method of  claim 31 , wherein the target DNA sequence binding region is between -10 and -35 regions of the regulatory element. 
     
     
         33 . The method of  claim 31 , wherein the target DNA sequence is a -10 region or a -35 region. 
     
     
         34 . The method of  claim 31 , wherein the target DNA sequence is in a eukaryotic regulatory element. 
     
     
         35 . The method of  claim 34 , wherein the target DNA sequence in the eukaryotic regulatory element is identified in a yeast two-hybrid assay. 
     
     
         36 . The method of  claim 1 , the position of the target DNA sequence recruits RNA polymerase. 
     
     
         37 . The method of  claim 1 , wherein the position of the target DNA sequence in the regulatory element is selected from: at the -10 or -35 region of the regulatory element, in the UP-region of the regulatory element, upstream of the -35 site, between the -10 and -35 sites. between the -10 and transcriptional start site, overlapping the transcriptional start site, and overlapping an activator binding site. 
     
     
         38 . The method of  claim 1 , wherein the sequence-specific DNA-binding polypeptide comprises a modification that results in recruitment of RNA polymerase to DNA bound by the sequence-specific DNA-binding polypeptide. 
     
     
         39 . The method of  claim 38 , wherein the modification is the addition of the C-terminal VP 16 sequence. 
     
     
         40 . The method of  claim 1 , wherein the orthogonal set is determined by identifying a set of sequence-specific DNA-binding polypeptides that do not bind to each other's target DNA sequences. 
     
     
         41 . The method of  claim 40 , wherein the designing cognate sequence-specific DNA binding polypeptide-target DNA sequence pairs comprises maximizing the size of the set of orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs. 
     
     
         42 . The method of  claim 40 , wherein the identifying comprises using a bioinformatic model built using empirical DNA binding data. 
     
     
         43 . The method of  claim 42 , wherein the bioinformatic model maximizes the diversity between target DNA sequences in the set of orthogonal-sequence-specific DNA binding polypeptide-target sequence pairs. 
     
     
         44 . The method of  claim 42 , wherein a graph partitioning algorithm is used to identify the maximum orthogonal set. 
     
     
         45 . The method of  claim 44 , wherein edges of the set are weighted by sequence entropy calculated by the set of DNA binding sequences to which two target DNA sequences bind. 
     
     
         46 . The method of  claim 2 , wherein the repressors are TetR homologues, zinc finger proteins, or Tal effectors. 
     
     
         47 . The method of  claim 46 , wherein the TetR homolgues are AcrR, AmtR, ArpA, BM3R1, BarA, Bell, EthR, FarA, HapR, HIyIIR, IcaR, LmrA, LuxT, McbR, MphR, MtrR, MtrR, Ph1F, PsrA, QacR, ScbR, SmcR, SmeT, TetR, TtgR, Ty1P, UidR, VarR. 
     
     
         48 . The method of  claim 2 , wherein the transcriptional activators are sigma factors. 
     
     
         49 . The method of  claim 1 , wherein the genetic circuit is determined using a logic minimisation algorithm. 
     
     
         50 . The method of  claim 49 , wherein the logic minimisation algorithm is ESPRESSO. 
     
     
         51 . The method of  claim 1 , wherein the genetic circuit is determined using a hardware descriptive language. 
     
     
         52 . The method of  claim 51 , wherein the hardware descriptive language is VHDL or Verilog. 
     
     
         53 . The method of  claim 1 , wherein the genetic circuit is a combination of logic gates. 
     
     
         54 . The method of  claim 53 , wherein the logic gates are selected from the group consisting of AND, NAND, NOR, OR, NOT, XOR, EQUALS, AND, IMPLIES, and ANDN gates. 
     
     
         55 . The method of  claim 54 , wherein the NOR gates comprise a transcriptional repressors and a transcriptional repressor target DNA sequence. 
     
     
         56 . The method of  claim 54 , wherein the AND gates comprises a sigma factor and a sigma factor target DNA sequence. 
     
     
         57 . The method of  claim 56 , wherein the sigma factor is a chimeric sigma factor comprising a first and second domain wherein the first and second domains are from two different sigma factors, wherein the first domain binds to a -10 region of a regulatory element and the second domain binds to a -35 region of a regulatory element. 
     
     
         58 . The method of  claim 2 , wherein the RNA polymerase is substantially identical to T7 RNA polymerase (RNAP). 
     
     
         59 . The method of  claim 2 , wherein the set of orthogonal pairs comprises at least two or more different RNA polymerases substantially identical to T7 RNA polymerase (RNAP). 
     
     
         60 . The method of  claim 58 , wherein the T7 RNAP has been modified from its native form to reduce toxicity to a heterologous organism. 
     
     
         61 . The method of  claim 60 , wherein the modification includes one or more of addition of an N-terminal Lon protease tag, a GTG start codon, and/or an R632S mutation. 
     
     
         62 . The method of  claim 2 , further comprising mutating T7 RNAP to generate an orthogonal set of polypeptides substantially identical to T7 RNAP that bind to different DNA sequences. 
     
     
         63 . The method of  claim 60  or  62 , wherein the polypeptides comprise a loop corresponding to the loop between 745 and 761 of T7 RNAP, wherein the loop is mutated to the sequence of a homologous phage polymerase. 
     
     
         64 . The method of  claim 58 , wherein the polymerase is from T3, K1F, or N4. 
     
     
         65 . The method of  claim 58 , wherein a cognate target DNA sequence is created by mutating at least one nucleotide of a T7 RNAP dependent promoter between nucleotides -13 and -18 
     
     
         66 . The method of  claim 58 , wherein a cognate target DNA sequence comprises a DNA binding sequence for T3, K1F, or N4 phage polymerase. 
     
     
         67 . The method of  claim 58 , wherein strength of the cognate target DNA sequence has been modified by mutating the nucleotides between -4 and -8. 
     
     
         68 . The method of  claim 58 , comprising generating a library of promoters with different strengths by recombining defined sequences between -13 and -18 with defined sequences between -4 and -8 of a DNA binding sequence for T7, T3, K1F, or N4 phage polymerase. 
     
     
         69 . The method of  claim 2 , wherein the transcriptional activator requires a second chaperone polypeptide to be bound to the activator to generate transcriptional activity. 
     
     
         70 . The method of  claim 69 , wherein the transcriptional activator is substantially identical to InvF (from  Salmonella typhimurium ), MxiE (from  Shigella flexneri ), or ExsA (from  Pseudomonas aeriginosa ). 
     
     
         71 . The method of  claim 69  or  70 , wherein the chaperone is substantially similar to SicA (from  Salmonella typhimuriwn ), IpgC (from  Shigella flexneri ), or ExsC (from  Pseudomonas aeriginosa ) 
     
     
         72 . The method of  claim 69 , wherein the transcriptional activator and chaperone are used to construct an AND gate. 
     
     
         73 . The method of  claim 72 , wherein one promoter serves as an input controls the expression of the activator and a second promoter that serves as an input controls the expression of the chaperone. 
     
     
         74 . A method of generating a library of orthogonal sigma factors, the method comprising
 generating a library of polynucleotides encoding chimeric sigma factors, wherein the chimeric sigma factors comprise a domain from at least two different sigma factors, wherein each of the domains bind to the -10 or -35 region of a regulatory element; and   expressing chimeric sigma factors from the library of polynucleotides, thereby generating a library of chimeric sigma factors.   
     
     
         75 . A host cell comprising a heterologous genetic circuit comprising at least two orthogonal sequence-specific DNA binding polypeptides, wherein the genetic circuit is a combination of logic gates. 
     
     
         76 . The host cell of  claim 75 , wherein the logic gates are selected from the group consisting of AND, NAND, NOR, OR NOT, XOR, EQUALS, AND, IMPLIES, and ANDN gates. 
     
     
         77 . The host cell of  claim 76 , wherein the NOR gates comprise a transcriptional repressors and a transcriptional repressor target DNA sequence. 
     
     
         78 . The host cell of  claim 76 , wherein the AND gates comprises a sigma factor and a sigma factor target DNA sequence. 
     
     
         79 . The host cell of  claim 78 , wherein the sigma factor is a chimeric sigma factor comprising a first and second domain wherein the first and second domains are from two different sigma factors, wherein the first domain binds to a -10 region of a regulatory element and the second domain binds to a -35 region of a regulatory element. 
     
     
         80 . The host cell of  claim 76 , wherein the at least two sequence-specific DNA binding polypeptides are selected from the group consisting of transcription factors, transcriptional activators, RNA polymerases, and transcriptional repressors. 
     
     
         81 . The host cell of  claim 76 , wherein the at least two sequence-specific DNA binding polypeptides are transcriptional activators. 
     
     
         82 . The host cell of  claim 76 , wherein the at least two sequence-specific DNA binding polypeptides are RNA polymerases. 
     
     
         83 . The host cell of  claim 76 , wherein the at least two sequence-specific DNA binding polypeptides are transcriptional repressors. 
     
     
         84 . The host cell of  claim 75 , wherein the logic gates comprise a regulatory element, wherein the regulatory element comprises a target DNA sequence bound by one of the sequence-specific DNA binding polypeptides and wherein the position of the target DNA sequence in the regulatory element is selected from: at the -10 or -35 region of the regulatory element, in the UP-region of the regulatory element, upstream of the -35 site, between the -10 and -35 sites. between the -10 and transcriptional start site, overlapping the transcriptional start site, and overlapping an activator binding site. 
     
     
         85 . The host cell of  claim 84 , wherein the at least two sequence-specific DNA binding polypeptides are selected from the group consisting of transcription factors, transcriptional activators, RNA polymerases, and transcriptional repressors. 
     
     
         86 . The host cell of  claim 75 , wherein the host cell is a prokaryotic host cell. 
     
     
         87 . The host cell of  claim 75 , wherein the gates are combined by having the output promoter of an upstream gate serve as the input promoter of a downstream gate. 
     
     
         88 . The host cell of  claim 87 , wherein a spacer sequence is included after the promoter that serves as a connection point between gates. 
     
     
         89 . The host cell of  claim 88 , wherein the spacer is encoded at the 5′-UTR of the mRNA encoding a transcription factor before the ribosome binding site. 
     
     
         90 . The host cell of  claim 88 , wherein the spacer forms a stem loop, is a native sequence from a metabolic pathway, or is from a 5′-UTR obtained from a phage. 
     
     
         91 . The host cell of  claim 90 , wherein the stem loop is a ribozyme. 
     
     
         92 . The host cell of  claim 91 , wherein the ribozyme is RiboJ. 
     
     
         93 . A non-transitory computer readable storage medium encoded with instructions, executable by a processor, for designing a host cell comprising a heterologous genetic circuit comprising at least two orthogonal sequence-specific DNA binding polypeptides, wherein the genetic circuit is a combination of logic gates, the instructions comprising:
 providing a set of sequence-specific DNA binding polypeptides;   optimizing expression of the polypeptides in a heterologous host cell;   identifying target DNA sequences to which the polypeptides bind;   generating synthetic transcriptional regulatory elements comprising at least one identified target DNA sequence, wherein the regulatory elements are responsive to a sequence-specific DNA binding polypeptide from the set of sequence-specific DNA binding polypeptides;   designing cognate sequence-specific DNA binding polypeptide-target DNA sequence pairs to generate one or more orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs;   designing a genetic circuit comprising a combination of logic gates, the logic gates comprising the one or more orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs.   
     
     
         94 . A computer product comprising a computer readable medium encoded with a plurality of instructions for controlling a computing system to perform an operation for designing a host cell comprising a heterologous genetic circuit comprising at least two orthogonal sequence-specific DNA binding polypeptides, wherein the genetic circuit is a combination of logic gates, the instructions comprising instructions for the steps of any of the methods of  claim 1 - 57 .