Difference between revisions of "Part:BBa K5124028"

 
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<partinfo>BBa_K5124028 short</partinfo>
 
<partinfo>BBa_K5124028 short</partinfo>
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===Usage and Biology===
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The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.
  
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Target from the bovine immune system with a T7 promoter for transcription
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In 2021 McLoughlin <i>et al.</i> published RNA-Seq data from cattle infected with bTB at several timepoints during the disease progression [1]. They identified 19 potential biomarkers that were present across the entire length of the infection time course. We would have liked to have tested all 19 sequences but after two rounds of the design-build-test-learn cycle (see our [https://2024.igem.wiki/exeter/engineering Wiki]) we focused on: 
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<br>CXCL8- chemokine ligand 8, involved in infection response and tissue injury.
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<br>FOSB- FBJ murine osteosarcoma viral oncogene homologue B, plays a role in regulating cell proliferation, differentiation and transformation.
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<br>NR4A1- nuclear receptor subfamily 4, group A, member 1, plays a role in inflammation and apoptosis.
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<br>PLAUR- plasminogen activator, urokinase receptor, a biomarker of inflammation.
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<br>RGS16- regulator of G-protein signalling 16, linked to several disease states.
  
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<!-- Add more about the biology of this part here
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This composite part contains a 5’ T7 promoter and a target sequence; a 100-nucleotide RNA sequence from the <i>Bos taurus</i> genome (BioProject accession number PRJNA450837). Once transcribed this sequence contains a 20-nucleotide sequence that is complimentary to the spacer sequence PLAUR (see basic part [https://parts.igem.org/Part:BBa_K5124022 BBa_K5124022] for details). This target sequence was used in our Cas13a assays as a mimic of bTB infected samples from cattle. In the final detection system, this part could be used as a positive control.
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===Usage and Biology===
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This sequence was synthesised by IDT as a composite part containing a 5’ T7 promoter ([https://parts.igem.org/Part:BBa_I719005 BBa_I719005]) and Type IIs compatible prefix and suffixes. As we needed the RNA sequence be identical to the <i>Bos taurus</i> genome, we were unable to remove the PstI restriction enzyme site. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker. 
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===Results===
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Put stuff hereeeeeeeee
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===References===
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1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.
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2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.
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3. Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.
  
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===Sequence and Features===
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K5124028 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5124028 SequenceAndFeatures</partinfo>
  

Revision as of 12:04, 22 September 2024


Cas13a PLAUR_target for transcription

Usage and Biology

The Exeter iGEM 2024 team are designing a rapid detection system for Bovine Tuberculosis (bTB) using CRISPR-Cas detection systems. The literature suggests that bTB infection in cattle can be detected by nucleic acid biomarkers in both blood [1] and tissue samples [2]. Therefore, there was potential to develop tests looking for both DNA and RNA biomarkers in infected cattle.

In 2021 McLoughlin et al. published RNA-Seq data from cattle infected with bTB at several timepoints during the disease progression [1]. They identified 19 potential biomarkers that were present across the entire length of the infection time course. We would have liked to have tested all 19 sequences but after two rounds of the design-build-test-learn cycle (see our Wiki) we focused on:
CXCL8- chemokine ligand 8, involved in infection response and tissue injury.
FOSB- FBJ murine osteosarcoma viral oncogene homologue B, plays a role in regulating cell proliferation, differentiation and transformation.
NR4A1- nuclear receptor subfamily 4, group A, member 1, plays a role in inflammation and apoptosis.
PLAUR- plasminogen activator, urokinase receptor, a biomarker of inflammation.
RGS16- regulator of G-protein signalling 16, linked to several disease states.

This composite part contains a 5’ T7 promoter and a target sequence; a 100-nucleotide RNA sequence from the Bos taurus genome (BioProject accession number PRJNA450837). Once transcribed this sequence contains a 20-nucleotide sequence that is complimentary to the spacer sequence PLAUR (see basic part BBa_K5124022 for details). This target sequence was used in our Cas13a assays as a mimic of bTB infected samples from cattle. In the final detection system, this part could be used as a positive control.

This sequence was synthesised by IDT as a composite part containing a 5’ T7 promoter (BBa_I719005) and Type IIs compatible prefix and suffixes. As we needed the RNA sequence be identical to the Bos taurus genome, we were unable to remove the PstI restriction enzyme site. The g-block was cloned into a high copy plasmid (origin of replication from pUC18 [3]) carrying an ampicillin selection marker.

Results

Put stuff hereeeeeeeee

References

1. McLoughlin KE, Correia CN, Browne JA, Magee DA, Nalpas NC, Rue-Albrecht K, et al. RNA-Seq Transcriptome Analysis of Peripheral Blood From Cattle Infected With Mycobacterium bovis Across an Experimental Time Course. Frontiers in Veterinary Science. 2021; 8:662002.

2. Taylor GM, Worth DR, Palmer S, Jahans K, Hewinson RG. Rapid detection of Mycobacterium bovis DNA in cattle lymph nodes with visible lesions using PCR. BMC Vet Res. 2007 Jun 13; 3:12.

3. Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct; 19(3):259-68.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 136
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 136
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 136
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 136
  • 1000
    COMPATIBLE WITH RFC[1000]