Berkeley LBL/Laina Notebook
From 2007.igem.org
Contents |
Introduction
A genomic library was constructed by partial digestion with EcoRI/BamHI and BgII/XhoI followed by ligation of all the fragments into EcoRI/XhoI and BgII/XhoI site of the cosmid vector pET29bEBBX. Oligonucleotide primers were synthesized by Intergrated DNA technologies (IDT),Inc, Coraville, IA. Nucleotide sequence analysis was analysed using Lasergene Megasoftware. Database search was done using Basic Local Alignment Search Tool (BLAST). Sequence Compilation Sequence Alignment. The presence of the photosynthetic gene cluster involved in the steps of the bacteriochlorophyll/chlorophyll biosynthesis pathway between Mg-chelation and formation of chlorophyllide (bchI, bchD, bchH, bchJ, bchM,bchE,bchL,bchN and bchB) were found by inspection of the ORFs (Xion et al).
Materials
Plasmids, Cells, Miniprep Kits, IPTG, Antibiotics.
Methods
Primer Design
Biobricking of bch-gene cluster and crtN gene.
Plasmids:
| pET29EBBX|pBR322|pHM6
Genes:
crtN PCR of crtN an Heliobacillus mobilis G/C content an annealing region crtN-F 40% BglII CrtN-R 50% BamHI and XhoI Primers crtN-F 5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’ crtN-R 3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’ bchB-F 5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA3' bchB-R 3'ttaGGATCCccgttcgccttggtttgacttact5' bchE-F 5'ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca3' bchE-R 3'gctagCTCGAGtattttcatcatgcctctcgt5' bchI-F 5'ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT3' bchI-R 3'ttaGGATCCtcggggctgagaaggcgggagca5' bchL-F 5'ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA3' bchL-R 3'ttaGGATCCttgggcagaaggtgtggaagca5' bchM-F 5'ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC3' bchM-R 3'ttaaGGATCCtctcttcggcttaatttccaacag5' bchN-F 5'accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac3' bchN-R 3'attaggatccTCATTCCAGCCACCCCGCTT5' PCR PCR of bch-? genes using - 20ng/ul pHM6 1ul pHM6 1ul dNTP 5ul primer mix 0.5ul enzyme-phusion polymerase 32.5ul H20 50ul total Digestions 6 ul bch insert 8ul pET29bEBBX 2ul ligase buffer 1ul ligase 3ul H20 20ul total Dephosphorylation 50 ul pBR3222 6 ul phosphatase buffer 1 ul phosphatase 3 ul H20 Incubate 30" -1hr run gel Ligations (3hrs @ R.T) ResultsSequencing Discussion |
