Team:Arizona State/Results/Data

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ASU Logo.png

E. coli

Construction of RSR+Leader Array

  • Gel results: Correct size for the RSR+Leader Array in the 6th well. Confirmed with DNASU sequencing.
ASU RSRLeader.jpg
  • Sequencing verification showed a 99% base pair match for "Seq1" and the MG1655 leader sequence, confirming the successful assembly of the array


Isolation of Cas genes

  • CasABCDE was unsuccessful, though we came close:
ASU 719 casABCDE.jpg
  • Cas3 was moderately successful, but unconfirmed via sequencing:
ASU 725 gel cas3.jpeg

Assembly of Leader+RSR into pRSF Duet

  • Assembled on 9/28/2011.

Poly-X Ligation

We devised a new assembly method, the Poly-X Ligation, for RSR construction and tested it on an RSR construct with GFP.

Poly-X RSR.jpg

Contingency Test

  • Standardized competent cells
  • Plate results:
Absorbance Conditions
96 Well Plate Type Corning Costar
Well Surface Area 0.32 cm2
Sample Volume 0.1 cm3
Path Distance 0.3125 cm
Pre-Competency Preparation
Culture Raw Absorbance / Recorded OD600* Mean OD600
BL21 L+Seq1 0.216 0.302
0.965 		0.309
0.987 		0.976
1655 L+Seq1 0.343
1.098 		0.351
1.122 		0.354
1.131 		1.117
BL21 Leader 0.259
0.830 		0.271
0.867 		0.272
0.871 		0.856
1655 Leader 0.224
0.716 		0.215
0.688 		0.215
0.689 		0.697
BL21 GFP 0.317
1.014 		0.342
1.096 		0.321
1.026 		1.045
1655 GFP 0.127
0.406 		0.137
0.438 		0.125
0.400 		0.415
trash 0.046
0.148 		0.047
0.149 		0.046
0.148 		0.148
trash 0.046
0.148 		0.047
0.149 		0.047
0.149 		0.148
Amp blank 0.039
0.123 		0.041
0.131 		0.040
0.127 		0.040
0.128 		0.127
Kan blank 0.040
0.127 		0.040
0.128 		0.041
0.131 		0.040
0.128 		0.129

*Recorded OD600 = raw absorbance * 3.2

Competency Prep Dilution
(1/100th dilution, grown for 2 hours)
Culture Raw Absorbances / Recorded OD600 Mean OD600
BL21 L+Seq1 0.081
0.260 		0.079
0.252 		0.083
0.265 		0.259
1655 L+Seq1 0.077
0.245 		0.078
0.250 		0.085
0.272 		0.256
BL21 Leader 0.068
0.218 		0.072
0.229 		0.077
0.246 		0.231
1655 Leader 0.060
0.193 		0.061
0.195 		0.062
0.198 		0.196
BL21 GFP 0.054
0.171 		0.054
0.172 		0.055
0.175 		0.173
1655 GFP 0.047
0.149 		0.048
0.153 		0.048
0.152 		0.151
Transformation Efficiency Results
E. Coli Strain Initial Plasmid Introduced Plasmid Antibiotic selection DNA Quantity Used (ng) # of Colonies Weighted Colony Count**
MG1655 GFP Construct in pRSF Leader in pIDT Amp 140 21 138.742
MG1655 GFP Construct in pRSF Leader+Seq1 in pIDT Amp 147 31 204.810
MG1655 Leader in pIDT GFP Construct in pRSF Kan 143.4 23 117.635
MG1655 Leader+Seq1 in pIDT GFP Construct in pRSF Kan 143.4 1000 3909.508
BL21 GFP Construct in pRSF Leader in pIDT Amp 140 0 0.000
BL21 GFP Construct in pRSF Leader+Seq1 in pIDT Amp 147 2 11.574
BL21 Leader in pIDT GFP Construct in pRSF Kan 143.4 14 60.512
BL21 Leader+Seq1 in pIDT GFP Construct in pRSF Kan 143.4 9 35.186

**Weighted Colony Count = (# of Colonies)/(mean OD600 of competency prep)

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

Isolation of cmr genes

  • Our construct requires the isolation of 6 genes, Cmr1-6, that are all located on a single locus.
  • Gel results (CMR success):
ASU 716 cmr success.jpg

Assembly into pRSF Duet

L. innocua

Construction of RSR Array

  • This construction of the RSR array involved the ligation of two customized BioBricks (sequences), which can be done simultaneously using this protocol.

Cas gene

  • The L. innocua CRISPR system utilizes three CRISPR-associated genes: 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.

Assembly into pRSF Duet

  • Cas9+tracrRNA array has been ligated into pRSF Duet
ASU Listeria Gel 9-28.PNG

Other

  • Construction of constitutive GFP plasmid by ligation of Bba_E0840 with Bba_(promoter)
  • Success was marked by green colonies
ASU gfpconstruct.jpg

Contact Us

Arizona State University

ECG 334, PO BOX 9709

Tempe, Arizona 85287

Email

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