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]. 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).
  
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Fwd primer: 5’ AATTTAATACGACTCACTATAGGGAAACAAACAAA<b>GA</b>CCTTGCTGTCACCGGAATG 3’
  
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===Usage in biology===
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Rv primer: 5’ CACACAGGAAACAGCTATGACCATG 3’
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Sensing small molecules is the foundation of many applications, ranging from disease diagnosis, prognosis, or treatment to detecting small pollutants in the environment. Synthetic genetic switches are a promising tool to detect and quantify small molecules. Conventional synthetic biology-based biosensors are based on transcription factors. However, transcription factors are not easily reprogrammable with respect to ligand selectivity.  Antibody-based biosensors can be easily developed into new sensing capabilities. Unfortunately, these types of sensors are not suitable to detect low molecular weight compounds. RNA-based biosensors are an interesting alternative since they present a remarkable flexibility to be engineered into sensing a wide range of analytes with high sensitivity and selectivity. In particular, aptazymes, ligand regulated self-cleaving ribozymes, are of special  interest as cleavage of the aptazyme can be coupled to regulated gene expression in vivo or in vitro. Moreover, newly developed methods such as DRIVER (de novo rapid in vitro evolution of RNA biosensors) enable rapid, automated, and multiplexed engineering of aptazymes sequences to diverse ligands.
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The TU Delft 2021 team provides the iGEM community with a novel genetic switch construct (BBa_K3806016) in which an aptazyme sequence is fused to the <i>lacZ</i> reporter gene to convert a ligand concentration to a colorimetric read-out. The BBa_K3806016 contains a theophylline-binding aptazyme sequence [1], yet this part is designed to be modular and serve as a template to engineer other ligand-specific genetic circuits by swapping the aptazyme domain. This part was successfully expressed in a cell-free system, and its biosensing performance was compared to BBa_K3806014, showing an improvement in the dynamic range and time-response capabilities.
 
  
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[[File:T--TUDelft--missmatch.jpg|500px|center|]]
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<html><i><b>Fig. 1 Secondary structure of the transcribed products from (A) BBa_K3806014 (cRBS) (B) BBa_K3806016 (semi-cRBS).</b></i></html> The RBS is shaded in magenta and the two modified nucleotides of the antisense strand are pointed with a black arrow.
  
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For more information in this design, visit the <html><a href="https://2021.igem.org/Team:TUDelft/Design">Design</a></html> page of the TU Delft 2021 team.
  
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==References==
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===Source===
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[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.
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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
    INCOMPATIBLE WITH RFC[12]
    Unknown
  • 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.