A frame-shifted gene, which rescued its function by non-natural start codons and its application in constructing synthetic gene circuits
Autor: | Kathakali Sarkar, Sayak Mukhopadhyay, Deepro Bonnerjee, Rajkamal Srivastava, Sangram Bagh |
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Jazyk: | angličtina |
Rok vydání: | 2019 |
Předmět: | |
Zdroj: | Journal of Biological Engineering, Vol 13, Iss 1, Pp 1-10 (2019) |
Druh dokumentu: | article |
ISSN: | 1754-1611 12313734 |
DOI: | 10.1186/s13036-019-0151-x |
Popis: | Abstract Background Frame-shifted genes results in non-functional peptides. Because of this complete loss of function, frame-shifted genes have never been used in constructing synthetic gene circuits. Results Here we report that the function of gene circuits is rescued by a frame-shifted gene, which functions by translating from a non-natural start codon. We report a single nucleotide deletion mutation that developed in the λ-repressor cI within a synthetic genetic NOT gate in Escherichia coli during growth and through this mutation, a non-functional synthetic gene circuit became functional. This mutation resulted in a frame-shifted cI, which showed effective functionality among genetic NOT-gates in Escherichia coli with high regulatory ranges (> 300) and Hill coefficient (> 6.5). The cI worked over a large range of relative copy numbers between the frame-shifted gene and its target promoter. These properties make this frame-shifted gene an excellent candidate for building synthetic gene circuits. We hypothesized a new operating mechanism and showed evidence that frame-shifted cI was translated from non-natural start codon. We have engineered and tested a series of NOT gates made from a library of cI genes, each of which starts from a different codon within the first several amino acids of the frame-shifted cI. It is found that one form with start codon ACA, starting from the 3rd codon had similar repression behavior as the whole frame-shifted gene. We demonstrated synthetic genetic NAND and NOR logic-gates with frame-shifted cI. This is the first report of synthetic-gene-circuits made from a frame-shifted gene. Conclusions This study inspires a new view on frame-shifted gene and may serve as a novel way of building and optimizing synthetic-gene-circuits. This work may also have significance in the understanding of non-directed evolution of synthetic genetic circuits. |
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