Difference between revisions of "Part:BBa K2974500"
Monicacho123 (Talk | contribs) |
|||
| Line 3: | Line 3: | ||
<partinfo>BBa_K2974500 short</partinfo> | <partinfo>BBa_K2974500 short</partinfo> | ||
| − | + | BBa_J23106 Toehold GFP is a construct developed by the 2019 Lambert iGEM team in order to be utilized as a biosensor. This part is designed to be used in conjunction with BBa_K2550001. From previous experience, the team was aware that a strong promoter such as BBa_J23100 from the Anderson Series of Promoters may produce overexpression or unintended transcription and expression of the reporter gene in a toehold construct. In order to improve upon these side effects, the team replaced the promoter from BBa_J23100 to BBa_J23106. In addition, the team replaced the LacZ reporter with eGFP to replace the color-based quantification system with a fluorescence-based quantification system. | |
| + | |||
| + | <strong>Descriptions</strong> | ||
| + | |||
| + | <strong>Results</strong> | ||
| + | |||
| + | The Medium Promoter Toehold GFP Biobrick (BBa_K2974500) was assembled into pSB3C5, a medium copy plasmid. After obtaining the BBa_J23106 promoter Toehold GFP gene block, the team digested the toehold insert using EcoRI-HF and PstI-HF and then confirmed the digest using gel electrophoresis. Furthermore, the team ligated the insert into a pSB3C5 vector digest and transformed the ligation with DH5α E.coli competent cells. The team then purified the DNA and sent it to sequencing, which came back successful. Sequencing results along with an additional gel confirmed the successful assembly of the toehold insert. | ||
<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Revision as of 12:33, 21 October 2019
Medium Promoter (BBa_J23106) Toehold LacZ
BBa_J23106 Toehold GFP is a construct developed by the 2019 Lambert iGEM team in order to be utilized as a biosensor. This part is designed to be used in conjunction with BBa_K2550001. From previous experience, the team was aware that a strong promoter such as BBa_J23100 from the Anderson Series of Promoters may produce overexpression or unintended transcription and expression of the reporter gene in a toehold construct. In order to improve upon these side effects, the team replaced the promoter from BBa_J23100 to BBa_J23106. In addition, the team replaced the LacZ reporter with eGFP to replace the color-based quantification system with a fluorescence-based quantification system.
Descriptions
Results
The Medium Promoter Toehold GFP Biobrick (BBa_K2974500) was assembled into pSB3C5, a medium copy plasmid. After obtaining the BBa_J23106 promoter Toehold GFP gene block, the team digested the toehold insert using EcoRI-HF and PstI-HF and then confirmed the digest using gel electrophoresis. Furthermore, the team ligated the insert into a pSB3C5 vector digest and transformed the ligation with DH5α E.coli competent cells. The team then purified the DNA and sent it to sequencing, which came back successful. Sequencing results along with an additional gel confirmed the successful assembly of the toehold insert.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]