IGEM:Cambridge/2008/Notebook/Voltage/BioBrick Manipulation

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=OsmY=
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=OsmY RBS=
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OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix.
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OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix, but in addition to have RBSs of 3 different strengths (B0030, B0031 and B0032) included in the suffix.
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After PCRing the DNA concentration was determined with the nanodrop, and determined to be 57.8, 47.8 and 58.8 ng/μL respectively.
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They were then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzymes. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. TOP10 cells were then transformed with the ligation product. The cells were then grown on chloramphenicol plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.
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They were then all cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. This then gave a volume ratio of 6.25μL to 0.4, 0.5 and 0.4μL for A,B and C. TOP10 cells were then transformed with the ligation product with highly competent TOP10 for extract C. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C and culture C was found to have grown colonies.
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Single colonies were then re PCRed using standard BioBrick protocol and also colonies were innoculated into 10ml LB with 35μg/mL chloramphenicol.
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=OsmY and RBS=
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==Primers==
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The RBS to be used is B0030 and the PCR product is used. OsmY plasmid is opened with Spe and RBS is cut with Xba and Spe. Ligation between the 2 is performed but founfd to be unsatisfactory, with the OsmY self ligating too readily. RBS is now cut with just Xba as this leaves it being a relatively large piece so more likely to ligate. In addition purification can be carried out - any pieces which are re-circularised have no insert. Now the whole is cut with Spe and then ligated, hopefully circularising the plasmid. A further modification is to use decreasing temperature, from 42-35°C, to cause accurate annealing of the sticky ends.
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OsmY Forward:        CTA TGA ATT CAT ATT CTA GAG CTG GCA CAG GAA CGT TAT CC                                     
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OsmY-B0030 Reverse:  CGC GCT GCA GCT CTA CTA GTT TTC TCC TCT TTA ATT TGT TAA ATA TAG A                          
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OsmY-B0031 Reverse:  CTC TCT GCA GCT CTA CTA GTG GTT TCC TGT GTG ATT GTT AAA TAT AGA T             
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OsmY-B0032 Reverse:  CTC TCT GCA GCT CTA CTA GTC TTT CCT GTG TGA TTG TTA AAT ATA GAT CA           
=Kdp=
=Kdp=
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Kdp was PCRed out of the chromosome of MG1655, in two different tubes. The primers were designed to have the BioBrick prefix and suffix.
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Kdp was PCRed out of the chromosome of MG1655. The primers were designed to have the BioBrick prefix and suffix.
-
After PCRing the DNA concentration was determined with the nanodrop, and determined to be 127.5 and 70.5ng/μL.
+
It was then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. As Kdp is 4.4kb and the vector is 4kb approximatley the 1.1:1 ratio of mass was used (Kdp:pSB4C5). TOP10 cells were then transformed with the ligation product. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C.
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They were both then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. As Kdp is 4.4kb and the vector is 4kb approximatley the 1.1:1 ratio of mass was used (Kdp:pSB4C5), giving a volume ratio of 6.25μL to 3μL and 5.1μL. TOP10 cells were then transformed with the ligation product with highly competent TOP10 for extract 1. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C and neither culture was found to have grown colonies.
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==Primers==
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KDP Forward:  ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT               
 +
KDP Reverse:  CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T           
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=InFusion=
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InFusion primers were designed for both OsmY-RBS and KDP as well as the terminator containing plasmid B1002. These primers were designed to give the backbone with the terminator still attached.  
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==Primers==
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Forward:  ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT               
 +
Reverse:  CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T

Revision as of 05:39, 28 October 2008

Contents

OsmY RBS

OsmY was PCRed out of the biobricks which contained it - BBa_J45996 (A) , BBa_J45995 (B) and BBa_J45250 (C). The primers were designed to have the BioBrick prefix and suffix, but in addition to have RBSs of 3 different strengths (B0030, B0031 and B0032) included in the suffix. They were then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzymes. This was done at a 16:1 ratio of masses as OsmY is about 250bp and pSB4C5 is 4kbp. TOP10 cells were then transformed with the ligation product. The cells were then grown on chloramphenicol plates at 37°C and single colonies were then PCRed using standard BioBrick protocol.

Primers

OsmY Forward: CTA TGA ATT CAT ATT CTA GAG CTG GCA CAG GAA CGT TAT CC OsmY-B0030 Reverse: CGC GCT GCA GCT CTA CTA GTT TTC TCC TCT TTA ATT TGT TAA ATA TAG A OsmY-B0031 Reverse: CTC TCT GCA GCT CTA CTA GTG GTT TCC TGT GTG ATT GTT AAA TAT AGA T OsmY-B0032 Reverse: CTC TCT GCA GCT CTA CTA GTC TTT CCT GTG TGA TTG TTA AAT ATA GAT CA

Kdp

Kdp was PCRed out of the chromosome of MG1655. The primers were designed to have the BioBrick prefix and suffix. It was then cut with EcoRI and PstI and ligated into pSB4C5, which had been cut with the same enzyme. As Kdp is 4.4kb and the vector is 4kb approximatley the 1.1:1 ratio of mass was used (Kdp:pSB4C5). TOP10 cells were then transformed with the ligation product. The cells were then plated onto 35μg/mL chloramphenicol and grown at 37°C.

Primers

KDP Forward: ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT KDP Reverse: CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T

InFusion

InFusion primers were designed for both OsmY-RBS and KDP as well as the terminator containing plasmid B1002. These primers were designed to give the backbone with the terminator still attached.

Primers

Forward: ATA TGA ATT CAT ATT CTA GAT GAG TGC AGG CGT GAT AAC CGG CGT ATT Reverse: CTC TCT GCA GCT CTA CTA GTT TAT TCA TCA AGT TTA TCC AGC GCC AGA T

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