Berkeley LBL/Laina Notebook

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Biobricking of bch-gene cluster and crtN gene.  
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== 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).
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Gene:
+
== '''Materials''' ==
 +
Plasmids, Cells, Miniprep Kits, IPTG, Antibiotics.
 +
 
 +
== '''Methods''' ==
 +
'''Primer Design'''
 +
 
 +
Biobricking of bch-gene cluster and crtN gene.
 +
 
 +
Plasmids:
 +
{| border="0" cellspacing="8px" cellpadding="15" width="80%"
 +
||[[pET29EBBX]]|[[pBR322]]|[[pHM6]]
 +
Genes:
{| border="0" cellspacing="8px" cellpadding="15" width="80%"
{| border="0" cellspacing="8px" cellpadding="15" width="80%"
|[[bchB]]
|[[bchB]]
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crtN  
crtN  
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PCR of crtN on Heliobacillus mobilis  
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PCR of crtN an Heliobacillus mobilis  
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G/C content on annealing region
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G/C content an annealing region
crtN-F    40% BglII
crtN-F    40% BglII
Line 20: Line 33:
Primers
Primers
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crtN-F    5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
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crtN-R    3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’
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bchB-F    5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA3'
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bchB-R    3'ttaGGATCCccgttcgccttggtttgacttact5'
 +
bchE-F    5'ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca3'
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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'''
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  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)
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crtN-F    cloning of crtN  5’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt3’
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== '''Results''' ==
 +
 +
'''Sequencing'''
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crtN-R  cloning of crtN  5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
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== '''Discussion''' ==
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bch B|3'ttaGGATCCccgttcgccttggtttgacttact
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Latest revision as of 21:24, 3 November 2008

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:

bchB bchE bchI bchL bchM bchN [[crtNgene]]

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)

Results

Sequencing

Discussion

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