Team:Arizona State/Results/Data

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

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• 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

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• Assembly into pRSF Duet

• Cas9+tracrRNA array has been ligated into pRSF Duet

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Other

• Construction of constitutive GFP plasmid by ligation of Bba_E0840 with Bba_(promoter)

• Success was marked by green colonies