Team:Heidelberg/Templates/DelH overview22

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===Results===
===Results===
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===Vector Maps and Primers===
===Vector Maps and Primers===

Revision as of 11:43, 4 October 2013

Results

Colonies Alignment File Sequence
III F7 File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b F7.clustal.txt Mutation
III F9 File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b F9.clustal.txt Mutation
III G3 File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b G3.clustal.txt Mutation
III G4 File:Heidelberg Sequencing Result pHM04 VF2 colony DH10b G4.clustal.txt Mutation


Vector Maps and Primers

Strategy A: weak Promotor, weak RBS
Expression of the possibly toxic DelH module on pFS02 is minimized by usage of a weak promoter: BBa_J23114 as well as a weak RBS: BBa_0032.

Vector map of the weak promotor and weak RBS DelH plasmid pFS02 (pSB6A1_BBa_J23114_BBa_B0032_DelH) Gibson plasmid.
Identifier Order date Note Sequence
FS_78:BB_HPLC_rev 26-09-2013 Gibson-Primer rev, amplify the Backbone pSB6A1 introducing the
RBS BBa_B0032 and the promotor BBa_J23114 and
creating an overlap to the first fragment of
DelH amplified with primer DN_11		 GATTTGGCGCAGGCGGCCACGG
TCCATCTAGTACTTTCCTGTGTGACTCTA
GAGCTAGCATTGTACCTAGGACTGAGCT
AGCCATAAACTCTAGAAGCGGCCGCGAATTC
FS_84:BB_HPLC_fw 26-09-2013 Gibson-Primer fw to amplify first fragment of DelH introducing
the RBS BBa_B0032 and creating an overlap to
primer FS_85 thereby partially introducing the
promotor BBa_J23114		 GCTCAGTCCTAGGTACAATGCTAGC
TCTAGAGTCACACAGGAAAGTACTAGATGGA
CCGTGGCCGCCTGCG
FS_85:BB_HPLC_rev 26-09-2013 Gibson-Primer rev to amplify the Backbone pSB6A1, partially
introducing the promotor BBa_B0032 with overlap to primer
FS_84 and therefore the promotor BBa_J23114,
it creates an overlap to the beginning of DelH		 GCGATTTGGCGCAGGCGGCCACGGT
CCATCTAGTATTTCTCCTCTTTC


Strategy B: ccdB Construct
Selection for mutated and thus truncated DelH sequences will be minimized by using a ccdB strategy. DelH will be integrated in a pSB4K5 backbone flanked by KpnI and BamHI sites (pFS02). The pSB6A1 backbone will be assembled with a ccdB cassette flanked by KpnI and BamHI sites (pFS03). The final DelH plasmid will be assembled by restriction - ligation of pFS02 and pFS03 to pFS05.

Identifier Order date Note Sequence
FS_85:BB_HPLC_rev 26-09-2013 Gibson-Primer rev to amplify the Backbone pSB6A1, partially introducing the
promotor BBa_B0032 with overlap to primer FS_84 and therefore
the promotor BBa_J23114, it creates an overlap to the beginning of DelH		 GCGATTTGGCGCAGGCGGCCACGGTCCATCTAGTATTTCTCCTCTTTC
FS_86:BB_HPLC_rev 26-09-2013 Gibson-Primer rev to amplify the Backbone pSB4K5 without any promotor
introducing a KpnI cutting site for restriction cloning, creates an overlap
to DelH and will be used for the ccdB strategy		 GGCGATTTGGCGCAGGCGGCCACGGTCCATGTACTTCGAGTCACTAAGGGCTAAC
FS_87:BB_HPLC_fw 26-09-2013 Gibson-Primer fw to amplify the Backbone pSB6A1 introducing a BamHI
cutting site for restriction cloning and creating an overlap to the last
fragment of DelH		 CGCTGGAGTACGCGCTGGACTGAGATCCCAGGCATCAAATAAAACG
FS_90:ccdB_HPLC_fw 26-09-2013 Gibson-Primer fw to amplify the ccdB cassette from the template pDonor
Plasmid introducing a KpnI cutting site for restriction cloning, creates an
overlap to the promotor BBa_J23114 and will be used for
the ccdB strategy		 CTCAGTCCTAGGTACAATGCTAGCTCTAGAGTCACACAGGAAAGCAGTAC
ACTGGCTGTGTATAAGGGAG
FS_93:ccdB_HPLC_rev 26-09-2013 Gibson-Primer rev to amplify the ccdB cassette from the template pDonor
Plasmid introducing a BamHI cutting site for restriction cloning,
creates an overlap to the backbone pSB6A1 and will be used for
the ccdB strategy		 GTTCACCGACAAACAACAGATGATCCGCGTGGATCCGGCTTAC
FS_94:BB_HPLC_fw 26-09-2013 Primer fw to amplify the backbone pSB6A1, will be used for the ccdB strategy ATCTGTTGTTTGTCGGTGAACGC
Vector map of the DelH plasmid without promoter in pSB4K5 pFS03 (pSB4K5min_KpnI_DelH_BamHI) Gibson plasmid.
Vector map of the pSB6A1 backbone with ccdB cassette pFS04 (pSB6A1_BBa_J23114_BBa_B0032_ccdB_cassette) Gibson plasmid.
Vector map of the final DelH plasmid in pSB6A1 pFS05 (DelH_final_pSB6A1_BBa_J23114_BBa_B0032_KpnI_DelH_BamHI) Gibson plasmid.


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