Difference between revisions of "Part:BBa K2230000"
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Cloning: Firstly, the elements of upstream and downstream recombination sites were replicated from gDNA of Lactobacillus acidophilus and put onto pSB1C3 as a L. acidophilus recombination vector, named pLBA169, at EcoRI and PstI locations. Secondly, to generate as a standard BioBrick part and assembly vector, the part of Promoter CP29-RBS-aeBlue (BBa_K1033280) was cut by EcoRI and PstI and assembled with the vector. Finally, because there’s a SpeI recognition sequence within the 169dn element, site-directed mutagenesis was performed to change a nucleotide to leave a single SpeI site in the BioBrick suffix region. (See the flow chart and data below). | Cloning: Firstly, the elements of upstream and downstream recombination sites were replicated from gDNA of Lactobacillus acidophilus and put onto pSB1C3 as a L. acidophilus recombination vector, named pLBA169, at EcoRI and PstI locations. Secondly, to generate as a standard BioBrick part and assembly vector, the part of Promoter CP29-RBS-aeBlue (BBa_K1033280) was cut by EcoRI and PstI and assembled with the vector. Finally, because there’s a SpeI recognition sequence within the 169dn element, site-directed mutagenesis was performed to change a nucleotide to leave a single SpeI site in the BioBrick suffix region. (See the flow chart and data below). | ||
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Revision as of 04:09, 25 October 2017
CP29-RBS-aeBlue/pLBA169
A vector can be used to transform Lactobacillus acidophilus by chromosomal homologous recombination at the downstream location of slpA (LBA0169), which encodes a surface layer protein A. The aeBlue gene, which is driven by the wide host range and high constitutive promoter CP29, acts as a reporter. The transformed L. acidophilus will express blue color proteins.
Cloning: Firstly, the elements of upstream and downstream recombination sites were replicated from gDNA of Lactobacillus acidophilus and put onto pSB1C3 as a L. acidophilus recombination vector, named pLBA169, at EcoRI and PstI locations. Secondly, to generate as a standard BioBrick part and assembly vector, the part of Promoter CP29-RBS-aeBlue (BBa_K1033280) was cut by EcoRI and PstI and assembled with the vector. Finally, because there’s a SpeI recognition sequence within the 169dn element, site-directed mutagenesis was performed to change a nucleotide to leave a single SpeI site in the BioBrick suffix region. (See the flow chart and data below).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3356
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 3362 - 21INCOMPATIBLE WITH RFC[21]Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 3356
Illegal XhoI site found at 1642
Illegal XhoI site found at 2534 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found at 3356
Illegal suffix found at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found at 3356
Plasmid lacks a suffix.
Illegal XbaI site found at 3371
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000INCOMPATIBLE WITH RFC[1000]Plasmid lacks a prefix.
Plasmid lacks a suffix.