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Home
iGEM 2011 Home
Project
Introduction
CRISPR
E. coli
B. halodurans
L. innocua
Software
Future
References
Lab
Team
Photos
Protocols
Safety
Acknowledgements
Results
Data
BioBricks
Notebook
April/May
June
July
August
September
Cas PCR Log
Sequence Information
Other Documents
Human Practices
Events
Collaborations
Exploring Synthetic Biology
Outreach in Practice
E. coli
Construction of RSR+Leader Array
Gel results:
Image
Sequencing verification showed a 99% base pair match for "Seq1" and the MG1655 leader sequence, confirming the successful assembly of the array
File:Screenshot
Isolation of Cas genes
Unsuccessful but we learned a lot!
Assembly of Leader+RSR into pRSF Duet
Gel results pending TONIGHT
Contingency Test
Standardized competent cells
Plate results
B. halodurans
Construction of RSR Array
We constructed a 1x Repeat-Spacer-Repeat array by ligating our "RA" (Spacer-Repeat) sequence to our "RB" (Repeat) sequence
Gel results: (do we have sequencing confirmation?)
Isolation of cmr genes
Our construct requires the isolation of 6 genes, Cmr1-6, that are all located on a single locus.
Gel results
Image
Assembly into pRSF Duet
L. innocua
Construction of RSR Array
This construction of the RSR array involved the ligation of two customized BioBricks (link), which can be done simultaneously using this protocol (link).
Gel results for 1x
Image
Cas gene
The L. innocua CRISPR system utilizes three CRISPR-associated genes (link?): Cas1, Cas2, and Cas9.
For our streamlined construct, we needed to isolate only Cas9, which is approximately 4kb in length.
We PCR amplified Cas9 with the adjacent trans-encoded CRIPSR RNA region (tracrRNA), which is necessary for the formation of mature CRISPR RNA.
Gel results
Image
Assembly into pRSF Duet
Cas9+tracrRNA array has been ligated into pRSF Duet
Image
Other
Construction of constitutive GFP plasmid by ligation of Bba_E0840 with Bba_(promoter)
Success was marked by green colonies
Image