Difference between revisions of "Part:BBa K3799026"

(→‎Cloning and characterization)
(→‎Cloning and characterization)
 
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'''Cloning:'''<br>
 
'''Cloning:'''<br>
 
From IDT, gene blocks with a 6x-His-tag, tobacco etch virus protease cut site, and restriction enzyme cut sites on the 5' and 3' end for NcoI and BlpI, respectively was oredered. After digesting the gene segments with restriction enzymes, they were ligated to the pET-52b expression vector. The assembled plasmid was transformed into a C41 pLysS cell line. Colonies were grown on selective media to screen the cells containing transformed plasmids containing Csm6 gene. Colony PCR was performed to make sure that the transformed plasmids were correct in insertion and orientation.
 
From IDT, gene blocks with a 6x-His-tag, tobacco etch virus protease cut site, and restriction enzyme cut sites on the 5' and 3' end for NcoI and BlpI, respectively was oredered. After digesting the gene segments with restriction enzymes, they were ligated to the pET-52b expression vector. The assembled plasmid was transformed into a C41 pLysS cell line. Colonies were grown on selective media to screen the cells containing transformed plasmids containing Csm6 gene. Colony PCR was performed to make sure that the transformed plasmids were correct in insertion and orientation.
−
[[Image:T--UCSC--100512-TI-678Csm6.png | thumb | center | 800 px |Colony PCR of EiCsm6 and TtCsm6]]
+
[[Image:T--UCSC--100512-TI-678Csm6.png | thumb | center | 400 px |Colony PCR of EiCsm6 and TtCsm6]]
−
[[Image:T--UCSC--100512-TI-678Csm602.jpg | thumb | center | 800 px |PCR of plasmids with EiCsm6 and TtCsm6 with the same primers used in colony PCR]]
+
As the results of the colony PCR for TtCsm6 is a bit unclear, both the teams decided to run a PCR amplification of the isolated plasmid from the positive colonies with the primers same as that of the colony PCR. This would confirm the presence of insert in the isolated plasmids.
 +
[[Image:T--UCSC--100512-TI-678Csm602.jpg | thumb | center | 400 px |PCR of plasmids with EiCsm6 and TtCsm6 with the same primers used in colony PCR]]
 +
As we can see, both the isolated plasmids have the intended lengths of insert reflecting successful cloning and plasmid isolation. UCSC Igem team 2021 used those colonies to purify protein by stimulating expression overnight with IPTG, followed by cell lysis and sonication, but the final results were not available before the wiki freeze.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 02:13, 22 October 2021


Type III-A CRISPR-Cas effector ribonuclease---Csm6

Csm6 is a non-specific endoribonuclease that cleaves single-stranded RNA upon activation by cyclic adenylates or linear adenine homopolymers terminated with a 2'3'-cyclic phosphate.

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]

Usage and Biology

Description - EiCsm6 is obtained from Enterococcus italicus, and belongs to the type III CRISPR systems. Csm6 is mainly activated by cyclic oligoadenylate second messengers by binding to their CARF domain. This activates the HEPN domain of the Csm6 for collateral RNA degradation. The CARF domain then degrades the cyclic adenylate activator (cA6) which functions as an off switch to regulate the activity of the CRISPR enzyme. These mechanisms facilitate rapid invader clearance and ensures proper termination to limit self toxicity and suicide of the bacteria.

EiCsm6 is coupled with the enzyme LwaCas13a(BBa_K2323004) to create the detector system SHERLOCKv2. The overhangs that are produced by the cleavage of RNA by Cas13a produces 2’-3’ cyclic phosphate hex adenylate residues that activate Csm6. This activation results in the collateral cleavage of RNA by both Cas13a and Csm6.


Cloning and characterization

Cloning:
From IDT, gene blocks with a 6x-His-tag, tobacco etch virus protease cut site, and restriction enzyme cut sites on the 5' and 3' end for NcoI and BlpI, respectively was oredered. After digesting the gene segments with restriction enzymes, they were ligated to the pET-52b expression vector. The assembled plasmid was transformed into a C41 pLysS cell line. Colonies were grown on selective media to screen the cells containing transformed plasmids containing Csm6 gene. Colony PCR was performed to make sure that the transformed plasmids were correct in insertion and orientation.

Colony PCR of EiCsm6 and TtCsm6

As the results of the colony PCR for TtCsm6 is a bit unclear, both the teams decided to run a PCR amplification of the isolated plasmid from the positive colonies with the primers same as that of the colony PCR. This would confirm the presence of insert in the isolated plasmids.

PCR of plasmids with EiCsm6 and TtCsm6 with the same primers used in colony PCR

As we can see, both the isolated plasmids have the intended lengths of insert reflecting successful cloning and plasmid isolation. UCSC Igem team 2021 used those colonies to purify protein by stimulating expression overnight with IPTG, followed by cell lysis and sonication, but the final results were not available before the wiki freeze.