Part:BBa_K4705006
Rate limiting enzyme of protoporphyrin IX with Temperature sensitive switch
This part contains the coding gene of the thermo-sensitive transcription factor TCI under the constitutive promoter J23119 In 2019, UCAS-China developed a collection of thermosensitive parts with high-performance, versatility and robustness. Based on TCI transcription factor family and TlpA family, we collected five TCI and TlpA mutants and used sfGFP as reporter to build some heat-inducible ON-switches, which can open gene expression under high temperature. To characterize them quantitatively, we first characterized the performance of them by flow cytometer in Top10 strain.
For target gene, we choose protoporphyrin IX as the small molecule metabolite to degrade the antibody. So we search the synthetic pathway of protoporphyrin IX in website metacyc and find the rate limiting enzyme-----glutamyl-tRNA reductase in the synthetic pathway of protoporphyrin IX. Then we use database uniprot to find the enzyme in Lactobacillus plantarum L168. We find that In prokaryotes, the genes for enzymes are determined by HemA . So we decide to overexpress the HemA gene in Lactobacillus plantarum.As we have got the amino acid sequence of enzyme,we use DNAfasta database to get the DNA sequence of this enzyme. At last we use NCBI to blast the sequence with our Lactobacillus plantarum L168 and lock the sequence we need.
and we choose Temperature sensitive switch to control the gene. the promoter is controlled by TCL protein which is mentioned in our previous design
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 631
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 576
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