Difference between revisions of "Part:BBa K4041005"
 
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4041005 short</partinfo>
 
<partinfo>BBa_K4041005 short</partinfo>
−
 
−
<br>Template for T7 transcription of crRNA for Vibrio pathogenic gene marker. Need to be used with a XbaI overnight clean cut or PCR for the transcription to end at break point, to generate a short crRNA instead of transcription doesn't end. Designed on 100% conserved region.
 
−
 
−
 
−
 
 
https://static.igem.org/mediawiki/parts/0/08/HKIS--crRNA_dem.png
 
https://static.igem.org/mediawiki/parts/0/08/HKIS--crRNA_dem.png
−
<br>Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation.
+
<br>Figure 1: Demonstration for a 2 oligo anneal system that is similar to a plasmid double stranded transcription system. The upper strand is annealed to the “T7 promoter” primer for reverse transcription. Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation.
  
  
Line 18: Line 13:
 
https://static.igem.org/mediawiki/parts/a/a9/HKIS--crRNAbuff.png
 
https://static.igem.org/mediawiki/parts/a/a9/HKIS--crRNAbuff.png
 
<br>crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly.
 
<br>crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly.
 +
 +
<table style="border-color:383737;" bgcolor="white">
 +
           
 +
                <tr>
 +
                    <td>The following crRNA were proved to work in this block in in Vibrio</td>
 +
               
 +
                </tr>
 +
<tr>
 +
                    <td>TDH forward (crRNA1)</td>
 +
<td>ACTGTGAACATTAATGATAA</td>
 +
               
 +
                </tr>
 +
 +
<tr>
 +
                    <td>vcgC forward (crRNA3)</td>
 +
<td>ATAGCACTAATGTGTCATCT</td>
 +
               
 +
                </tr>
 +
<tr>
 +
                    <td>vcgC reverse (crRNA4, with PAM)</td>
 +
<td>GCGGAGACGAGATCGCTATC</td>
 +
               
 +
                </tr>
 +
             
 +
<tr>
 +
                    <td>ctxA Forward (crRNA5)</td>
 +
<td>TGATCATGCAAGAGGAACTC</td>
 +
               
 +
                </tr>
 +
 +
<tr>
 +
                    <td>crRNA 2 (high background) and crRNA 6 (no cleavage signal) is not functional from design, and is discarded</td>
 +
 +
               
 +
                </tr>
 +
               
 +
        </table>
 +
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 20:16, 21 October 2021


TDH cas12a crRNA transcription device (crRNA1) HKIS--crRNA_dem.png
Figure 1: Demonstration for a 2 oligo anneal system that is similar to a plasmid double stranded transcription system. The upper strand is annealed to the “T7 promoter” primer for reverse transcription. Need to be used with a T7 promoter based short RNA transcription kit, like MEGAshortscript RNA transcription kit, followed by purification, like spin columns or lithium ion precipitation.




HKIS--ureapage.png
15% UREA Page (Run in room temperature with 80V), indicating that the crRNA is successfully transcribed



HKIS--crRNAbuff.png
crRNA used with single stranded template, showcasing that the crRNA best work with 1X NEB2.1 buffer and functions to cleave reporter very rapidly.


The following crRNA were proved to work in this block in in Vibrio
TDH forward (crRNA1) ACTGTGAACATTAATGATAA
vcgC forward (crRNA3) ATAGCACTAATGTGTCATCT
vcgC reverse (crRNA4, with PAM) GCGGAGACGAGATCGCTATC
ctxA Forward (crRNA5) TGATCATGCAAGAGGAACTC
crRNA 2 (high background) and crRNA 6 (no cleavage signal) is not functional from design, and is discarded


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]