Team:Arizona State/Results/Data

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Revision as of 03:43, 29 September 2011

E. coli

Construction of RSR+Leader Array

Gel results: Correct size for the RSR+Leader Array in the 6th well. Confirmed with [http://sequencing.biodesign.asu.edu/ DNASU] sequencing.

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:

Cas3 was moderately successful, but unconfirmed via sequencing:

Assembly of Leader+RSR into pRSF Duet

Gel results pending TONIGHT

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.

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):

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

Other

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

Success was marked by green colonies

@@ Line 5: / Line 5: @@
 ===Construction of RSR+Leader Array===
 :*Gel results: Correct size for the RSR+Leader Array in the 6th well. Confirmed with [http://sequencing.biodesign.asu.edu/ DNASU] sequencing.
-[[Image: ASU_RSRLeader.jpg|400px]]
+[[Image: ASU_RSRLeader.jpg|400px|center]]
 :*Sequencing verification showed a 99% base pair match for "Seq1" and the MG1655 leader sequence, confirming the successful assembly of the array
@@ Line 11: / Line 11: @@
 ===Isolation of Cas genes===
 :*CasABCDE was unsuccessful, though we came close:
-[[Image: ASU_719_casABCDE.jpg|400px]]
+[[Image: ASU_719_casABCDE.jpg|400px|center]]
 :*Cas3 was moderately successful, but unconfirmed via sequencing:
-[[Image: ASU_725_gel_cas3.jpeg|400px]]
+[[Image: ASU_725_gel_cas3.jpeg|400px|center]]
 ===Assembly of Leader+RSR into pRSF Duet===
@@ Line 20: / Line 20: @@
 ===[https://2011.igem.org/Team:Arizona_State/Lab/Protocols/Assembly#Poly 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.
-[[image: Poly-X_RSR.jpg|400px]]
+[[image: Poly-X_RSR.jpg|400px|center]]
 ===Contingency Test===
@@ Line 33: / Line 33: @@
 ===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
+:*Gel results (CMR success):
-[[Image: ASU_716_cmr_success.jpg|400px|CMR Success]]
+[[Image: ASU_716_cmr_success.jpg|400px|center]]
 ===Assembly into pRSF Duet===
@@ Line 44: / Line 43: @@
 ==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).
+:*This construction of the RSR array involved the ligation of two customized BioBricks ([[Team:Arizona State/Notebook/Sequences|sequences]]), which can be done simultaneously using this [[Team:Arizona State/Lab/Protocols/Assembly|protocol]].
-:*Gel results for 1x
 ===Cas gene===
-:*The L. innocua CRISPR system utilizes three CRISPR-associated genes (link?): Cas1, Cas2, and Cas9.
+:*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.
-:*Gel results
 ===Assembly into pRSF Duet===
 :*Cas9+tracrRNA array has been ligated into pRSF Duet
-[[Image: ASU_Listeria_Gel_9-28.PNG|600px]]
+[[Image: ASU_Listeria_Gel_9-28.PNG|600px|center]]
 ==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]]
+[[Image: ASU_gfpconstruct.jpg|400px|center]]
 }}