Difference between revisions of "Part:BBa K3806016:Design"
 
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===Design Notes===
 
===Design Notes===
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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).
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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]. To obtain this part, <HTML><a href="https://parts.igem.org/Part:BBa_K3806014" target="_blank">BBa_K3806014</a></HTML> was PCR amplified using the primers listed below. 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’

Latest revision as of 22:33, 21 October 2021


Theophylline-binding aptazyme regulating lacZ expression (semi-cRBS). With T7 promoter.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3204
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2321
  • 1000
    COMPATIBLE 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]. To obtain this part, BBa_K3806014 was PCR amplified using the primers listed below. 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’


T--TUDelft--missmatch.jpg

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. [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.