Difference between revisions of "Part:BBa K2997000"
| Line 10: | Line 10: | ||
We amplified the tyrP gene and its promoter from E. coli MG1655 and cloning into pSB4A3. Then transformed the plasmid into DH5a and E. coli Nissle 1917, and extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. | We amplified the tyrP gene and its promoter from E. coli MG1655 and cloning into pSB4A3. Then transformed the plasmid into DH5a and E. coli Nissle 1917, and extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. | ||
[[File:T--NCKU Tainan--Result-TyrP-double digest 2.jpg]] | [[File:T--NCKU Tainan--Result-TyrP-double digest 2.jpg]] | ||
| − | + | ||
| − | + | ===TAL and TyrP Functional Assay=== | |
| − | + | We performed a functional test using n-octanol extraction method (https://2019.igem.org/Team:NCKU_Tainan/Protocols) | |
| + | , and with this assay, we can further improve the conversion of tyrosine into p-Coumaric acid by adding a tyrosine transporter (BBa_K2997000). As seen in figure., when BBa_K2997000 is added, the production of p-Coumaric acid is significantly higher than when BBa_K2997000 is not added. When tyrosine transporter is introduced into E. coli Nissle containing BBa_K2997009 and BBa_K2997010, conversion of tyrosine into p-Coumaric acid is increased by 1.44-fold and 1.31-fold respectively. | ||
| + | [[File:T--NCKU Tainan--Results tyrosine to pCA, RBS improve.png]] | ||
| + | |||
| + | |||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 14:19, 18 October 2019
Tyrosine transporter (tyrP)
Background
It’s the gene encode for tyrosine transporter that is involved in transporting tyrosine across the cytoplasmic membrane. Hence, we used PCR to amplify this gene from E. coli MG1655 and incorporated it to our E. coli, allowing it to take in more tyrosine.
Expression in E. coli
We amplified the tyrP gene and its promoter from E. coli MG1655 and cloning into pSB4A3. Then transformed the plasmid into DH5a and E. coli Nissle 1917, and extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. File:T--NCKU Tainan--Result-TyrP-double digest 2.jpg
TAL and TyrP Functional Assay
We performed a functional test using n-octanol extraction method (https://2019.igem.org/Team:NCKU_Tainan/Protocols) , and with this assay, we can further improve the conversion of tyrosine into p-Coumaric acid by adding a tyrosine transporter (BBa_K2997000). As seen in figure., when BBa_K2997000 is added, the production of p-Coumaric acid is significantly higher than when BBa_K2997000 is not added. When tyrosine transporter is introduced into E. coli Nissle containing BBa_K2997009 and BBa_K2997010, conversion of tyrosine into p-Coumaric acid is increased by 1.44-fold and 1.31-fold respectively. File:T--NCKU Tainan--Results tyrosine to pCA, RBS improve.png
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 278
- 1000COMPATIBLE WITH RFC[1000]