Difference between revisions of "Part:BBa K2797003:Design"
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===Design Notes=== | ===Design Notes=== | ||
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| + | [[Image: mneon.png|thumb|right|400px| '''A simulated Gibson Assembly of the mNeonGreen fluorescent protein sequence into the pSB1C3 vector backbone.''' As shown, the mNeonGreen slots into the regions in between the biobrick prefix and suffix, a region where the test device is usually present. The green regions show the mNeon green insert. The mNeongreen gene uses the promoter, RBS and terminator of the test device.]] | ||
Since the idea was to replace the GFPmut3b reporter gene in each of the test device pSB1C3 with the mNeonGreen construct, removal of the GFPmut3b coding region from each vector was required - this was done using 2-step PCR. Six reverse primers complimentary to each of the test device RBS and their varying promoter regions, and 1 a single forward primer to bind at the beginning of the terminator were utilised in 2-step PCR to linearise the respective pSB1C3 vectors - removing GFPmut3b. The resulting amplification was treated with DpnI. | Since the idea was to replace the GFPmut3b reporter gene in each of the test device pSB1C3 with the mNeonGreen construct, removal of the GFPmut3b coding region from each vector was required - this was done using 2-step PCR. Six reverse primers complimentary to each of the test device RBS and their varying promoter regions, and 1 a single forward primer to bind at the beginning of the terminator were utilised in 2-step PCR to linearise the respective pSB1C3 vectors - removing GFPmut3b. The resulting amplification was treated with DpnI. | ||
The mNeonGreen sequence was codon optimised for expression in E. coli DH5-alpha using Benchling. Further to this, Gibson ends were designed using NEBuilder for cloning into the linearised pSB1C3 and added to the 3' and 5' ends of the mNeonGreen sequence. The mNeonGreen sequence was synthesised by IDT. The sequence was cloned into each pSB1C3 vector via Gibson Assembly. | The mNeonGreen sequence was codon optimised for expression in E. coli DH5-alpha using Benchling. Further to this, Gibson ends were designed using NEBuilder for cloning into the linearised pSB1C3 and added to the 3' and 5' ends of the mNeonGreen sequence. The mNeonGreen sequence was synthesised by IDT. The sequence was cloned into each pSB1C3 vector via Gibson Assembly. | ||
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===Source=== | ===Source=== | ||
Latest revision as of 12:50, 10 October 2018
mNeonGreen
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 39
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Since the idea was to replace the GFPmut3b reporter gene in each of the test device pSB1C3 with the mNeonGreen construct, removal of the GFPmut3b coding region from each vector was required - this was done using 2-step PCR. Six reverse primers complimentary to each of the test device RBS and their varying promoter regions, and 1 a single forward primer to bind at the beginning of the terminator were utilised in 2-step PCR to linearise the respective pSB1C3 vectors - removing GFPmut3b. The resulting amplification was treated with DpnI.
The mNeonGreen sequence was codon optimised for expression in E. coli DH5-alpha using Benchling. Further to this, Gibson ends were designed using NEBuilder for cloning into the linearised pSB1C3 and added to the 3' and 5' ends of the mNeonGreen sequence. The mNeonGreen sequence was synthesised by IDT. The sequence was cloned into each pSB1C3 vector via Gibson Assembly.
Source
Sequence from Allele Biotechnology