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E. coli
Construction of RSR+Leader Array
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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
- CasABCDE was unsuccessful, though we came close:
- Cas3 was moderately successful, but unconfirmed via sequencing:
Assembly of Leader+RSR into pRSF Duet
- Gel results pending TONIGHT
Contingency Test
- Standardized competent cells
- Plate results:
Absorbance Conditions
96 Well Plate Type
| Corning Costar
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Well Surface Area
| 0.32 cm2
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Sample Volume
| 0.1 cm3
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Path Distance
| 0.3125 cm
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Pre-Competency Preparation
Culture
| Raw Absorbance / Recorded OD600*
| Mean OD600
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BL21 L+Seq1 | 0.216 | 0.302 0.965 | 0.309 0.987 | | 0.976
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1655 L+Seq1 | 0.343 1.098 | 0.351 1.122 | 0.354 1.131 | | 1.117
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BL21 Leader | 0.259 0.830 | 0.271 0.867 | 0.272 0.871 | | 0.856
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1655 Leader | 0.224 0.716 | 0.215 0.688 | 0.215 0.689 | | 0.697
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BL21 GFP | 0.317 1.014 | 0.342 1.096 | 0.321 1.026 | | 1.045
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1655 GFP | 0.127 0.406 | 0.137 0.438 | 0.125 0.400 | | 0.415
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trash | 0.046 0.148 | 0.047 0.149 | 0.046 0.148 | | 0.148
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trash | 0.046 0.148 | 0.047 0.149 | 0.047 0.149 | | 0.148
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Amp blank | 0.039 0.123 | 0.041 0.131 | 0.040 0.127 | 0.040 0.128 | 0.127
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Kan blank | 0.040 0.127 | 0.040 0.128 | 0.041 0.131 | 0.040 0.128 | 0.129
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*Recorded OD600 = raw absorbance * 3.2
Competency Prep Dilution (1/100th dilution, grown for 2 hours)
Culture
| Raw Absorbances / Recorded OD600
| Mean OD600
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BL21 L+Seq1 | 0.081 0.260 | 0.079 0.252 | 0.083 0.265 | 0.259
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1655 L+Seq1 | 0.077 0.245 | 0.078 0.250 | 0.085 0.272 | 0.256
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BL21 Leader | 0.068 0.218 | 0.072 0.229 | 0.077 0.246 | 0.231
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1655 Leader | 0.060 0.193 | 0.061 0.195 | 0.062 0.198 | 0.196
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BL21 GFP | 0.054 0.171 | 0.054 0.172 | 0.055 0.175 | 0.173
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1655 GFP | 0.047 0.149 | 0.048 0.153 | 0.048 0.152 | 0.151
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Transformation Efficiency Results
E. Coli Strain
| Initial Plasmid
| Introduced Plasmid
| Antibiotic selection
| DNA Quantity Used (ng)
| # of Colonies
| Weighted Colony Count**
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MG1655 | GFP Construct in pRSF | Leader in pIDT | Amp | 140 | 21 | 138.742
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MG1655 | GFP Construct in pRSF | Leader+Seq1 in pIDT | Amp | 147 | 31 | 204.810
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MG1655 | Leader in pIDT | GFP Construct in pRSF | Kan | 143.4 | 23 | 117.635
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MG1655 | Leader+Seq1 in pIDT | GFP Construct in pRSF | Kan | 143.4 | 1000 | 3909.508
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BL21 | GFP Construct in pRSF | Leader in pIDT | Amp | 140 | 0 | 0.000
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BL21 | GFP Construct in pRSF | Leader+Seq1 in pIDT | Amp | 147 | 2 | 11.574
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BL21 | Leader in pIDT | GFP Construct in pRSF | Kan | 143.4 | 14 | 60.512
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BL21 | Leader+Seq1 in pIDT | GFP Construct in pRSF | Kan | 143.4 | 9 | 35.186
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**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
- 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
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
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