Difference between revisions of "Part:BBa K2927005:Experience"

 
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===Applications of BBa_K2927005===
 
===Applications of BBa_K2927005===
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<partinfo>BBa_K2927005 short</partinfo>
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<!-- Add more about the biology of this part here
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===Usage and Biology===
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'''UPF Barcelona ARIA Team experimental application:'''
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ARIA’s biosensors are based on two main elements: the endonuclease enzyme LbCas12a from Lachnospiraceae Bacterium, and the crRNAs designed specifically to target antibiotic resistance (AR) sequences. Once the two elements bind and the target is detected, a fluorescent signal is activated due to LbCas12a cis cleavage activity. However, these elements have been implemented on living E.Coli (BL21), and become active after lysing the cells and being put in contact with AR sequences. This has given rise to a new innovative approach: the in vivo – in vitro detection.
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To do so, the original plasmid employed for cloning Cas12 was taken from Addgene (<html><a href="https://www.addgene.org/113431/" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">https://www.addgene.org/113431/</a></html>), and it was optimized by deleting purification sites and unuseful sequences codifying for MBP (Maltose Binding Protein). Cas12 is expressed under the T7 promoter so that it is inducible with IPTG, and it has ampicillin resistance to allow for its selection.
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5 gRNAs were designed in total, each of them targeting the following resistance genes: Ampicillin, Chloramphenicol, Erythromycin, Kanamycin and Spectinomycin. Each gRNA is constituted by a common sequence of the structure DR + 24 bp-spacer + DR + L3S2P21 terminator. The spacer sequence is followed and preceded by a DR since Cas12 cuts the pre-crRNA 4 nucleotides upstream of the hairpin structures formed by the DR. This is important considering that Cas12a can process its own gRNAs (CRISPR RNAs) because of the dual RNase/DNase activity of Cas12a.
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We used the Substrate Nuclease Detection System (<html><a href="https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/dnasealert-detection-manual.pdf?sfvrsn=34133407_8" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/user-guide-manual/dnasealert-detection-manual.pdf?sfvrsn=34133407_8</a></html>) to perform detection and subsequent fluorescent measurements with the Plate Reader. To give a glimpse of the results we obtained, we present the analysis of a detection graph from the Chloramphenicol gRNA efficient construct . The fluorescence results for <html><a href="https://parts.igem.org/Part:BBa_K2927005" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K2927005</a></html> + <html><a href="https://parts.igem.org/Part:BBa_K3791021" target="_blank" onmouseover="this.style.color='#ff724a';" onmouseout="this.style.color='#ff9515';" style="color: #ff9515; text-decoration: none;">BBa_K3791021</a></html> (Cas12a +  gRNA for Chloramphenicol resistant gene) in Fig. 4 range between 26031 and 21607 RFU  and are coherent since the one with sample needs to be higher due to the gRNA - sample sequence match.
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The decrease in fluorescence signal is only 16.99% over 2 -hour time, which indicates a maintained stability of the signal. The biological significant negative control (without sample) produces a signal which is 38.1% lower in RFU than the one given by the biosensor with its corresponding sample. It ranges between 16118 and 14461 RFU. This is attributed to the nuclease activity released from the lysis process.  In both cases, the fluorescence is much higher than in the blank (negative control with water), a fact that is consistent. These results confirm our engineered biological system serves as a biosensor and accomplish the purpose for which it was created.
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[[Image:T--UPF_Barcelona--Description_graph_PoC_2and9_white.png |thumb|center|'''Fig 4. Fluorescence measurements for gRNA for Chloramphenicol resistant gene+Cas12a.'''
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<partinfo>BBa_K2927005 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K2927005 parameters</partinfo>
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===User Reviews===
 
===User Reviews===

Latest revision as of 14:31, 19 October 2021


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K2927005

LbCas12a



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1371
    Illegal BglII site found at 2108
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3022
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1435


User Reviews

UNIQ2bd2f333139a00dd-partinfo-00000002-QINU UNIQ2bd2f333139a00dd-partinfo-00000003-QINU