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Fig. 1 | Biotechnology for Biofuels

Fig. 1

From: Synthetic biology for evolutionary engineering: from perturbation of genotype to acquisition of desired phenotype

Fig. 1

Phenotypic perturbation methods. a, b Represent methods to alter transcriptional regulation. a Artificial TF and gTME library; transformation with artificial transcription factor or of global transcription machinery libraries that are generated by error-prone PCR results in phenotypic perturbation by unpredictable regulation changes. b CRISPRi/a; transformation with guide RNA (gRNA) plasmid array and dCas9 repressor/activator results in alter cellular regulations. c Perturbation of translational expression level through binding of sRNA and RNA binding protein. Expression of multiple sRNAs can repress multi-gene expression. d Representative two method for in vitro mutagenesis. A error-prone PCR introduce mutations during PCR and PCR with oligos harboring degenerate sequence diversify DNA sequence after being assembled. e MAGE technique introduces multiple mutations in genome through automation of iterative synthetic oligo recombineering. f ICE generate mutated DNA in vivo. A designed cassette composed of transposable element and target gene (CARGO) is transcribed and then reverse transcribed in an error-prone manner. The resulting cDNA is integrated back into the chromosome. d, g are methods to introduce mutations on target region. g EvolvR consists of a DNA polymerase fused with nCas9 which is recruited by gRNA into target region. After a DNA is nicked by the nCas9, the error-prone DNA polymerase performs error-prone strand displacement synthesis. h MutaT7 consists of a cytidine deaminase fused to T7 RNA polymerase. It is recruited to T7 promoter and mutations are loaded during transcription before transcription is terminated by terminator. i A plasmid carrying proof reading deficient DnaQ and factors conferring replication fidelity generates mutations during cell growth. j The pre-positioned loxPsym sites are recombined, inverted, and deleted by Cre induction and results various phenotypes

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