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{{:Team:Arizona_State/Templates/main|title=|content= __NOTOC__ ==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 [[Image: screenshot]] ===Isolation of Cas genes=== :*CasABCDE was unsuccessful, though we came close: [[Image: ASU_719_casABCDE.jpg|400px]] :*Cas3 was moderately successful, but unconfirmed via sequencing: [[Image: ASU_725_gel_cas3.jpeg|400px]] ===Assembly of Leader+RSR into pRSF Duet=== :*Gel results pending TONIGHT ===Poly-X Ligation=== [[image: Poly-X_RSR.jpg|400px]] ===Contingency Test=== :*Standardized competent cells :*Plate results: {{:Team:Arizona State/Templates/E coli contingency/table 1}} {{:Team:Arizona State/Templates/E coli contingency/table 2}} {{:Team:Arizona State/Templates/E coli contingency/table 3}} {{:Team:Arizona State/Templates/E coli contingency/table 4}} ==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: ASU_716_cmr_success.jpg|400px|CMR Success]] ===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: ASU_gfpconstruct.jpg|400px]] }}

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