Difference between revisions of "Part:BBa K3806016:Design"
| Line 7: | Line 7: | ||
===Design Notes=== | ===Design Notes=== | ||
| − | This biobrick represents the DNA template for the expression of the <i>lacZ</i> reporter gene in a T7 based cell-free expression system. Expression is regulated by a theophylline binding aptazyme [1]. The dsRNA sequence was PCR amplified from <HTML><a href="https://parts.igem.org/Part:BBa_K3806014" target="_blank">BBa_K3806014</a></HTML>. The forward primer used allowed for the change of the C and T (U in the aptazyme) nucleotides in the antisense strand into G and A, respectively, resulting in a partial mismatch to the RBS (Fig. 1). | + | This biobrick represents the DNA template for the expression of the <i>lacZ</i> reporter gene in a T7 based cell-free expression system. Expression is regulated by a theophylline binding aptazyme [1]. The dsRNA sequence was PCR amplified from <HTML><a href="https://parts.igem.org/Part:BBa_K3806014" target="_blank">BBa_K3806014</a></HTML>. The forward primer used allowed for the change of the C and T (U in the aptazyme) nucleotides in the antisense strand of the RBS into G and A, respectively, resulting in a partial mismatch to the RBS (Fig. 1). |
Fwd primer: 5’ AATTTAATACGACTCACTATAGGGAAACAAACAAA<b>GA</b>CCTTGCTGTCACCGGAATG 3’ | Fwd primer: 5’ AATTTAATACGACTCACTATAGGGAAACAAACAAA<b>GA</b>CCTTGCTGTCACCGGAATG 3’ | ||
Revision as of 00:50, 21 October 2021
Theophylline-binding aptazyme regulating lacZ expression (semi-cRBS). With T7 promoter.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3204
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2321
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
This biobrick represents the DNA template for the expression of the lacZ reporter gene in a T7 based cell-free expression system. Expression is regulated by a theophylline binding aptazyme [1]. The dsRNA sequence was PCR amplified from BBa_K3806014. The forward primer used allowed for the change of the C and T (U in the aptazyme) nucleotides in the antisense strand of the RBS into G and A, respectively, resulting in a partial mismatch to the RBS (Fig. 1).
Fwd primer: 5’ AATTTAATACGACTCACTATAGGGAAACAAACAAAGACCTTGCTGTCACCGGAATG 3’
Rv primer: 5’ CACACAGGAAACAGCTATGACCATG 3’
Fig. 1 Secondary structure of the transcribed products from (A) BBa_K3806014 (cRBS) (B) BBa_K3806016 (semi-cRBS). The RBS is shaded in magenta and the two modified nucleotides of the antisense strand are pointed with a black arrow.
For more information in this design, visit the Design page of the TU Delft 2021 team.
References
- [1] Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. and Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.