Team:Jilin China/NOTEBOOK

From 2014.igem.org

(Difference between revisions)
Line 283: Line 283:
<h3>Primer synthesis</h3>
<h3>Primer synthesis</h3>
<h4>Primer synthesis order o Comate Bioscience Co.,Ltd.</h4>
<h4>Primer synthesis order o Comate Bioscience Co.,Ltd.</h4>
-
<table border="1" cellspacing="0" cellpadding="0" style="float:center">
+
<table border="1" cellspacing="0" cellpadding="0" style="text-align:center">
   <tr>
   <tr>
     <td width="50" nowrap="nowrap"><p align="center"><strong>引物编号</strong><strong> </strong></p></td>
     <td width="50" nowrap="nowrap"><p align="center"><strong>引物编号</strong><strong> </strong></p></td>

Revision as of 19:44, 17 October 2014

Welcome!
Team Jilin China

NOTEBOOK

Synthesis and Characterisation of the Mlr Promoter

Research background

A novel pathway for degradation of the cyanobacterial heptapeptide hepatotoxin microcystin LR was identified in a newly isolated Sphingomonas sp. (Bourne et al. 1996 Appl. Environ. Microbiol. 62: 4086–4094). And the gene cluster involved in bacterial degradation of the cyanobacterial
toxin microcystin LR were be reported by David G. Bourne in 2001. The cloning and molecular characterisation of four genes from this Sphingomonas sp. that exist on a 5.8-kb genomic fragment and encode the three hydrolytic enzymes involved in this pathway together with a putative oligopeptide transporter. Situated immediately downstream of mlrA with the same direction of transcription is a gene mlrD, whose conceptual translation (MlrD, 442residues) shows significant sequence identity and similar potential transmembrane spanning regions to the PTR2family of oligopeptide transporters. A gene mlrB is situated downstream of the mlrA and mlrD genes, but transcribed in the opposite direction. The gene encodes the enzyme MlrB (402residues) which cleaves linear microcystin LR to a tetrapeptide degradation product. This enzyme belongs to the “penicillin-binding enzyme” family of active site serine hydrolases. The final gene in the cluster mlrC, is located upstream of the mlrA gene and is transcribed in the opposite direction. It codes for MlrC (507 residues) which mediates further peptidolytic degradation of the tetrapeptide. This protein shows significant sequence identity to a hypothetical protein from Streptomyces coelicolor. It is suspected to be a metallopeptidase based on inhibition by metal chelators. It is postulated on the basis of comparison with other microorganisms that the genes in this cluster may all be involved in cell wall peptidoglycan cycling and subsequently act fortuitously in hydrolysis of microcystin LR.
In this study, we  synthesized a RFP-promoter-GFP sequence to detect the relationship between MLR promoter and microcystin LR. This synthesized sequence using the promoter sequence between mlrA and mlrC, adding the red fluorescent protein sequence in the upstream to instead mlrC gene, and adding green fluorescent protein sequence in the downstream to instead mlrA gene.

 

Fig 1 Restriction map of the 5.8 kb of sequence data and localisation of genes involved in microcystin LR degradation.

methods

Synthetic sequence

    BamHI
GCCTGCAGctataaaaataaatgatgtctaccttctgttctttcatattgttcaacaattgtataatcttcattatgtgatgtaatatctaacttagcatctacataataatatcctggtaattgaactggttttttagccatataaattgatttaaattcaactaaataatgtcctccgtcctttaattttagagctttatgtgtttctccctttaaaaccccgtctcttggatataatctttctgtactagcttcccatcccattgtttttttttgcataactggtccgtctgatggaaaattaaccccaataaatttaactttataaataaaacaaccatcttgtaatgatgaatcttgtgtaactgtagcaactccaccatcttcaaaattcataactctttcccatttaaatccttctggaaaagataattttttataatctggaatatcagctggatgtttaacgtaaacctttgatccatattgaaattgtggtgataaaatatcccacgcaaatggtaatggtcctccttttgtaacctttaatttaactgtattatgtccttcataCggtctaccttctccttctccctcaatttcaaattcatgtccattaactgtaccttccattctaactttaaatctcataaattctgtaataacattttctgatgaagccatAacacgagtcttcggtttgctgttgtttgcaccagctgctgcatcttcaccccataaatcaaccgaacggacagtcaattcttgttgaccatcctgggcctatctgaaatgttctgctgacagaacgggagaattgaaccatgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttcggttatggtgttcaatgctttgcgagatacccagatcaCatgaaacagcatgactttttcaagagtgccatgcctgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataaGAATTCCG
EcoRI

Sequence analysis

 _Base Count : 1551 bp (489 A, 496 T, 314 C, 252 G)
 _Composition : 36% GC, 64% AT

Absent Sites
AarI AatII Acc65I AciI AclI AfeI AflII AgeI AhdI AleI ApaI ApaLI AscI AseI AsiSI AvaI BamHI BanI BanII BbvCI BceAI BciVI BclI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BsaBI BsaHI BsaWI BseMII BseRI BsgI BsiEI BsiHKAI BsiWI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI BsrDI BsrFI BssHII BstEII BstUI Bsu36I BtrI BtsI Cac8I ClaI DraIII EagI EarI EciI Eco53KI EcoNI EcoO109I EcoRV FauI FseI FspAI FspI HaeII HgaI HhaI HinP1I HindIII HpaII Hpy99I KasI KpnI MluI MmeI NaeI NarI NciI NdeI NgoMIV NheI NlaIV NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PfoI PmeI PpuMI PshAI PspOMI PspXI PstI PvuI RsrII SacI SacII SalI SanDI SbfI ScaI SexAI SfiI SfoI SgrAI SmaI SnaBI SpeI SphI SrfI SspI StuI TauI TspMI Tth111I XbaI XcmI XhoI XmaI XmnI ZraI
Unique Sites
Unique Sites
BamHI(3)
DdeI (92)
Esp3I (223)
SfcI (367)
BstXI (512) MslI (513)
BssSI (688) PleI (691) BbsI (693)
MwoI (714)
BpmI (851)
BsmFI (859)
Bsp1286I (895)
BtgI (991) MscI (994) Tsp45I (1006)
BsrGI (1101)
AflIII (1147) BsaAI (1148)
BstZ17I (1273)
HpaI (1312)
BtgZI (1391)
AsuII (1446) BstYI (1452) BsaI (1469) DrdI (1471)
SmlI (1486) BseYI (1504)
EcoRI (1543)

Building Block Design

The maximum allowable assembly oligo length is equal to the target assembly oligo length (60). This may cause some weird behavior, especially in terms of overlap melting temperature.

2 building blocks were generated.
Building Block mlrAC.1   789bp   1..789 
Left  - 5' GCCCTAGGCTATAAAAATAAATGATG 3'
Rght  - 5' ATAGGCCCAGGATGGTCAA 3'
RghtU - 5' ATAGGCCCAGGAUGGTCAA 3'
Sequence:
Assembly Oligos: average overlap Tm is 48°;average oligo length is 56bp.

GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAATTGTATAATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAATTGAACTGGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCCTTTAATTTTAGAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTACTAGCTTCCCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCCCAATAAATTTAACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCAACTCCACCATCTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAATCTGGAATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAAATATCCCACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTCATACGGTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTTCCATTCTAACTTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGAGTCTTCGGTTTGCTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGACAGTCAATTCTTGTTGACCATCCTGGGCCTAT
GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT      ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT      GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC          GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC       CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC           ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA          CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT    AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA      CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC     GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT          TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA            CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA                                
                                 AGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTG    GTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATT    TTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGG            AGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACG          GCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACAT    ACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGG        GGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGC          TAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGG            GAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGG            AGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGT    GACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACG            GGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA
CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGAT

CGGGATCCGATATTTTTATTTACTACAGATGGAAGACAAGAAAGTATAACAAGTTGTTAACATATTAGAAGTAATACACTACATTATAGATTGAATCGTAGATGTATTATTATAGGACCATTAACTTGACCAAAAAATCGGTATATTTAACTAAATTTAAGTTGATTTATTACAGGAGGCAGGAAATTAAAATCTCGAAATACACAAAGAGGGAAATTTTGGGGCAGAGAACCTATATTAGAAAGACATGATCGAAGGGTAGGGTAACAAAAAAAAACGTATTGACCAGGCAGACTACCTTTTAATTGGGGTTATTTAAATTGAAATATTTATTTTGTTGGTAGAACATTACTACTTAGAACACATTGACATCGTTGAGGTGGTAGAAGTTTTAAGTATTGAGAAAGGGTAAATTTAGGAAGACCTTTTCTATTAAAAAATATTAGACCTTATAGTCGACCTACAAATTGCATTTGGAAACTAGGTATAACTTTAACACCACTATTTTATAGGGTGCGTTTACCATTACCAGGAGGAAAACATTGGAAATTAAATTGACATAATACAGGAAGTATGCCAGATGGAAGAGGAAGAGGGAGTTAAAGTTTAAGTACAGGTAATTGACATGGAAGGTAAGATTGAAATTTAGAGTATTTAAGACATTATTGTAAAAGACTACTTCGGTATTGTGCTCAGAAGCCAAACGACAACAAACGTGGTCGACGACGTAGAAGTGGGGTATTTAGTTGGCTTGCCTGTCAGTTAAGAACAACTGGTAGGACCCGGATA    .
Building Block mlrAC.2   775bp   777..1551 
Left  - 5' ATCCTGGGCCTATCTGAAATG 3'
LeftU - 5' ATCCTGGGCCTAUCTGAAATG 3'
Rght  - 5' CGGAATTCTTATTTGTATAGTTCATCC 3'
Sequence:
Assembly Oligos: average overlap Tm is 49°;average oligo length is 53bp.
ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTGTTCAATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCTGAAGGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATACATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATAAGAATTCCG
ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCAT            AGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG       TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT          GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG            ATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGC          GGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA           GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT     AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA     TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTG     GGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGG         TTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCC           GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATG                               
                                CTTGCCCTCTTAACTTGGTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAG          AACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCC            TTGTATGCCTTTTGAATGGGAATTTAAATAAACGTGATGACCTTTTGATGGA            TGTGAACAGTGATGAAAGCCAATACCACAAGTTACGAAACGCTCTATGGGTC            TCGTACTGAAAAAGTTCTCACGGTACGGACTTCCAATACATGTCCTTTCTTGAT          TCTACTGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGG          CTTAGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTG         ATGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACC         ATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCT           TTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGAC         AGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAA           TCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATTCTTAAGGC
TAGGACCCGGATAGACTTTACAAGACGACTGTCTTGCCCTCTTAACTTGGTACTCATTTCCTCTTCTTGAAAAGTGACCTCAACAGGGTTAAGAACAACTTAATCTACCACTACAATTACCCGTGTTTAAAAGACAGTCACCTCTCCCACTTCCACTACGTTGTATGCCTTTTGAATGGGAATTTAAATAAACGTGATGACCTTTTGATGGACAAGGTACCGGTTGTGAACAGTGATGAAAGCCAATACCACAAGTTACGAAACGCTCTATGGGTCTAGTGTACTTTGTCGTACTGAAAAAGTTCTCACGGTACGGACTTCCAATACATGTCCTTTCTTGATATAAAAAGTTTCTACTGCCCTTGATGTTCTGTGCACGACTTCAGTTCAAACTTCCACTATGGGAACAATTATCTTAGCTCAATTTTCCATAACTAAAATTTCTTCTACCTTTGTAAGAACCTGTGTTTAACCTTATGTTGATATTGAGTGTGTTACATATGTAGTACCGTCTGTTTGTTTTCTTACCTTAGTTTCAATTGAAGTTTTAATCTGTGTTGTAACTTCTACCTTCGCAAGTTGATCGTCTGGTAATAGTTGTTTTATGAGGTTAACCGCTACCGGGACAGGAAAATGGTCTGTTGGTAATGGACAGGTGTGTTAGACGGGAAAGCTTTCTAGGGTTGCTTTTCTCTCTGGTGTACCAGGAAGAACTCAAACATTGTCGACGACCCTAATGTGTACCGTACCTACTTGATATGTTTATTCTTAAGGC 

Primer synthesis

Primer synthesis order o Comate Bioscience Co.,Ltd.

Top

引物编号

序列,(5' to 3')

碱基数(bp

合成总量(OD)

分装管数

纯化方式

AC101

GCCCTAGGCTATAAAAATAAATGATGTCTACCTTCTGTTCTTTCATATTGTTCAACAAT

59

1

1

PAGE

AC102

GTTAGATATTACATCACATAATGAAGATTATACAATTGTTGAACAATATGAAAGAACAGA

60

1

1

PAGE

AC103

ATCTTCATTATGTGATGTAATATCTAACTTAGCATCTACATAATAATATCCTGGTAAT

58

1

1

PAGE

AC104

TTAAATCAATTTATATGGCTAAAAAACCAGTTCAATTACCAGGATATTATTATGTAGATG

60

1

1

PAGE

AC105

GGTTTTTTAGCCATATAAATTGATTTAAATTCAACTAAATAATGTCCTCCGTCC

54

1

1

PAGE

AC106

GGGAGAAACACATAAAGCTCTAAAATTAAAGGACGGAGGACATTATTTAGTT

52

1

1

PAGE

AC107

GAGCTTTATGTGTTTCTCCCTTTAAAACCCCGTCTCTTGGATATAATCTTTCTGTAC

57

1

1

PAGE

AC108

GCAAAAAAAAACAATGGGATGGGAAGCTAGTACAGAAAGATTATATCCAAGAGA

54

1

1

PAGE

AC109

CCATCCCATTGTTTTTTTTTGCATAACTGGTCCGTCTGATGGAAAATTAACCC

53

1

1

PAGE

AC110

TACAAGATGGTTGTTTTATTTATAAAGTTAAATTTATTGGGGTTAATTTTCCATCAGACG

60

1

1

PAGE

AC111

ACTTTATAAATAAAACAACCATCTTGTAATGATGAATCTTGTGTAACTGTAGCA

54

1

1

PAGE

AC112

GGGAAAGAGTTATGAATTTTGAAGATGGTGGAGTTGCTACAGTTACACAAGATTCA

56

1

1

PAGE

AC113

CTTCAAAATTCATAACTCTTTCCCATTTAAATCCTTCTGGAAAAGATAATTTTTTATAAT

60

1

1

PAGE

AC114

CGTTAAACATCCAGCTGATATTCCAGATTATAAAAAATTATCTTTTCCAGAAGG

54

1

1

PAGE

AC115

AATATCAGCTGGATGTTTAACGTAAACCTTTGATCCATATTGAAATTGTGGTGATAAA

58

1

1

PAGE

AC116

GGACCATTACCATTTGCGTGGGATATTTTATCACCACAATTTCAATATGGAT

52

1

1

PAGE

AC117

CACGCAAATGGTAATGGTCCTCCTTTTGTAACCTTTAATTTAACTGTATTATGTCCTTC

59

1

1

PAGE

AC118

GGGAGAAGGAGAAGGTAGACCGTATGAAGGACATAATACAGTTAAATTAAAG

52

1

1

PAGE

AC119

GTCTACCTTCTCCTTCTCCCTCAATTTCAAATTCATGTCCATTAACTGTACCTT

54

1

1

PAGE

AC120

TGTTATTACAGAATTTATGAGATTTAAAGTTAGAATGGAAGGTACAGTTAATGGACATGA

60

1

1

PAGE

AC121

TTTAAATCTCATAAATTCTGTAATAACATTTTCTGATGAAGCCATAACACGA

52

1

1

PAGE

AC122

GCAGCTGGTGCAAACAACAGCAAACCGAAGACTCGTGTTATGGCTTCATCAG

52

1

1

PAGE

AC123

CTGTTGTTTGCACCAGCTGCTGCATCTTCACCCCATAAATCAACCGAACGGA

52

1

1

PAGE

AC124

ATAGGCCCAGGATGGTCAACAAGAATTGACTGTCCGTTCGGTTGATTTATGG

52

1

1

PAGE

AC201

ATCCTGGGCCTATCTGAAATGTTCTGCTGACAGAACGGGAGAATTGAACCAT

52

1

1

PAGE

AC202

GACAACTCCAGTGAAAAGTTCTTCTCCTTTACTCATGGTTCAATTCTCCCGTTC

54

1

1

PAGE

AC203

AGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGG

57

1

1

PAGE

AC204

CCCTCTCCACTGACAGAAAATTTGTGCCCATTAACATCACCATCTAATTCAA

52

1

1

PAGE

AC205

TTTTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTT

54

1

1

PAGE

AC206

AGGTAGTTTTCCAGTAGTGCAAATAAATTTAAGGGTAAGTTTTCCGTATGTT

52

1

1

PAGE

AC207

GCACTACTGGAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGG

52

1

1

PAGE

AC208

CTGGGTATCTCGCAAAGCATTGAACACCATAACCGAAAGTAGTGACAAGTGT

52

1

1

PAGE

AC209

ATGCTTTGCGAGATACCCAGATCACATGAAACAGCATGACTTTTTCAAGAGTGC

54

1

1

PAGE

AC210

TAGTTCTTTCCTGTACATAACCTTCAGGCATGGCACTCTTGAAAAAGTCATGCT

54

1

1

PAGE

AC211

GGTTATGTACAGGAAAGAACTATATTTTTCAAAGATGACGGGAACTACAAGA

52

1

1

PAGE

AC212

GGTATCACCTTCAAACTTGACTTCAGCACGTGTCTTGTAGTTCCCGTCATCT

52

1

1

PAGE

AC213

GTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTT

57

1

1

PAGE

AC214

GTGTCCAAGAATGTTTCCATCTTCTTTAAAATCAATACCTTTTAACTCGATTC

53

1

1

PAGE

AC215

AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA

57

1

1

PAGE

AC215

AGATGGAAACATTCTTGGACACAAATTGGAATACAACTATAACTCACACAATGTATA

57

1

1

PAGE

AC216

CCATTCTTTTGTTTGTCTGCCATGATGTATACATTGTGTGAGTTATAGTTGTA

53

1

1

PAGE

AC217

TGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAGACACAACATTG

57

1

1

PAGE

AC218

TCTGCTAGTTGAACGCTTCCATCTTCAATGTTGTGTCTAATTTTGAAGTTA

51

1

1

PAGE

AC219

GGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGG

53

1

1

PAGE

AC220

CAGGTAATGGTTGTCTGGTAAAAGGACAGGGCCATCGCCAATTGGAGTATTTT

53

1

1

PAGE

AC221

TTTACCAGACAACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCC

51

1

1

PAGE

AC222

AAGAAGGACCATGTGGTCTCTCTTTTCGTTGGGATCTTTCGAAAGGGCAGA

51

1

1

PAGE

AC223

GAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATG

51

1

1

PAGE

AC224

CGGAATTCTTATTTGTATAGTTCATCCATGCCATGTGTAATCCCAGCAGCT

51

1

1

PAGE

AC1

GCCCTAGGCTATAAAAATAAATGATG

26

1

1

PAGE

AC2

ATAGGCCCAGGATGGTCAA

19

1

1

PAGE

AC3

ATCCTGGGCCTATCTGAAATG

21

1

1

PAGE

AC4

CGGAATTCTTATTTGTATAGTTCATCC

27

1

1

PAGE

 

二、实验操作及结果分析

1、引物溶解

根据合成引物的分子量,计算需要添加的灭菌去离子水的量,将合成的引物稀释成10um/L备用。

2、引物混合(只有表格不一样,文字叙述是一样的)

合成的寡核苷酸呈粉末状,使用前需要10000rpm离心1min,使寡核苷酸干粉集中在管底,使用时小心开启瓶盖。
溶解寡核苷酸前核对合成报告单与引物标签上的OD数是否一致,并核对分装管数。根据每个引物的合成报告单计算出配制10umol/L浓度的寡核苷酸需要加入水的体积,加入去离子无菌水,室温放置2min,反复振荡助溶,再离心将溶液收集到管底。
每6条引物为一组,首末位引物取4ul,中间引物取1ul,加入新的EP管中,再补充灭菌去离子水至20ul,混匀备用。


组别

引物编号

吸取量

灭菌去离子水添加量

总体积

PCR50ul体系)吸取量

终浓度

A1

AC101

4

8

20

5ul

200nM

AC102

1

50nM

AC103

1

50nM

AC104

1

50nM

AC105

1

50nM

AC106

4

200nM

A2

AC107

4

8

20

5ul

200nM

AC108

1

50nM

AC109

1

50nM

AC110

1

50nM

AC111

1

50nM

AC112

4

200nM

A3

AC113

4

8

20

5ul

200nM

AC114

1

50nM

AC115

1

50nM

AC116

1

50nM

AC117

1

50nM

AC118

4

200nM

A4

AC119

4

8

20

5ul

200nM

AC120

1

50nM

AC121

1

50nM

AC122

1

50nM

AC123

1

50nM

AC124

4

200nM

A5

AC201

4

8

20

5ul

200nM

AC202

1

50nM

AC203

1

50nM

AC204

1

50nM

AC205

1

50nM

AC206

4

200nM

A6

AC207

4

8

20

5ul

200nM

AC208

1

50nM

AC209

1

50nM

AC210

1

50nM

AC211

1

50nM

AC212

4

200nM

A7

AC213

4

8

20

5ul

200nM

AC214

1

50nM

AC215

1

50nM

AC216

1

50nM

AC217

1

50nM

AC218

4

200nM

A8

AC219

4

8

20

5ul

200nM

AC220

1

50nM

AC221

1

50nM

AC222

1

50nM

AC223

1

50nM

AC224

4

200nM

3、DA-PCR

化学合成的单链寡核苷酸,每6条分为一组,通过DA-PCR拼接成为较长的双链中间片段(Block)。DA-PCR的反应体系组成为:
Mixed primer solutions                  5μl
Pfu DNA Polymerase 2.5U              0.5μl    
dNTP                                4μl
10×Pfu buffer(Mg2+)               5μl
灭菌超纯水                  补齐至  50μl
DA-PCR的反应程序为:
94℃         2min
94℃         30S
50℃         30S    25个循环
72℃         1min
72℃        10min
4℃          保存
End
DA-PCR拼接反应产物检测采用2%的琼脂糖凝胶电泳,其中
DA-PCR产物              5μl
10×Loading Buffer         0.6μl   混合均匀后点样
100bp Marker              5μl    直接点样
75V电泳1h。

4、OE-PCR

以DA-PCR拼接出来的的5条双链中间片段(Block)为模版,进一步利用OE-PCR进行连接,OE-PCR的反应体系组成为:
Block1                       1μl
Block2                       1μl
Block3                       1μl
Block4                       1μl
Block5                       1μl
Block6                       1μl
Block7                       1μl
Block8                       1μl
AC1                          1μl
AC4                          1μl
Pfu DNA Polymerase 2.5U       0.5μl
dNTP                        4μl
10×Pfu buffer(Mg2+)         5μl
          灭菌超纯水          补齐至  50μl
OE-PCR反应程序为:
94℃         2min
        94℃         30S
50℃         30S    20个循环
72℃        2min
72℃       10min
        4℃          保存
End
OE-PCR拼接得到的全长基因采用1%的琼脂糖凝胶电泳进行检测,其中
OE-PCR产物             5μl
10×Loading Buffer        0.6μl   混合均匀后点样
100bp Marker              5μl    直接点样
80V电泳1h。

5、亚克隆载体构建与测序

设计含有BlpⅠ酶切位点的扩增引物AC-B和含有SgrAⅠ酶切位点的扩增引物AC-S,以合成的RFP-promoter-GFP基因为模板,扩增制备外源基因;将其与pET32a载体经过BlpⅠ和SgrAⅠ于37℃双酶切3h后,16℃连接过夜,转化到E.coli BL21感受态细胞中,经蓝白筛选挑取白色菌落。
BlpⅠ和SgrAⅠ双酶切反应体系组成为:
Gene or plasmid                      10μl
BlpⅠ                               0.3μl
SgrAⅠ                             0.3μl
10×NEBuffer 2.1                      2μl
灭菌超纯水                  补齐至  20μl
连接反应体系组成为:

RFP-promoter-GFP 10μl
pET32a 3μl
T4 ligase 0.5μl
10×T4 ligase Buffer 2μl
灭菌超纯水 补齐至 20μl
E.coli BL21感受态细胞制备流程:
(1) E.coli BL21菌株37℃过夜培养以复苏菌种,取过夜培养的菌液600uL加入到30 mL LB培养基中,37℃,200 r/min振荡培养1-2 h,至OD600约为0.3-0.4左右。
(2) 无菌条件下,将30mL菌液转移至离心管中,冰上放置10 min,使培养物冷却至0℃。
(3) 在预冷4℃的离心机上以4000r/min离心10min,弃去上清,加入30mL预冷的0.1mol/L CaCl2溶液重悬沉淀,冰浴上放置10 min。
(4) 以4000r/min在4℃离心10min,弃去上清,每30mL初始培养物用1.0mL预冷的0.1mol/LCaCl2溶液重悬细胞沉淀,200μL/管分装。
热激转化过程:
(1) 取新鲜制备或者从-80℃冰箱中取出感受态细胞BL21悬液,冰浴解冻。
(2) 加入构建好的重组克隆载体质粒pSB1C3-A(含量不超过50ng,体积不超过10μl),轻轻摇匀,冰上放置30分钟后。
(3) 将该EP管放入预加温至42℃的水浴中,放置90s,快速转移到冰浴中,使细胞冷却1-2 min。
(4) 向EP管加入800 μL LB培养基,转移至37℃摇床上,150r/min,温育45 min以复苏菌株。
平板涂布及培养过程:
(1) 将37℃培养后的菌体低速离心(5000rpm、5min)浓缩为200μl,涂布到含有氨苄青霉素的LB平板上,设阳性对照。
(2) 将平板置于室温下至液体被完全吸收,倒置平皿,37℃过夜培养;
实验连接组:连接产物,氨苄青霉素板
阳性对照组: 氨苄青霉素板,以pSB1C3质粒代替DNA溶液, 其它操作与上面相同。此组正常情况下在含抗生素的LB平板上应产生大量菌落
阴性对照组:氨苄青霉素板,为连接时用水代替酶,其它与实验组相同。
空菌组:感受态细胞,无氨苄青霉素板
序列测定
在pET32a载体(图1)上,将BlpⅠ和SgrAⅠ之间的753bp片段使用新合成的RFP-promoter-GFP的1535bp的片段替换,将得到重组的pET-mlr promoter质粒(图2)。

图3 Map of pET-mlr promoter
挑取白色菌落,经液体培养后,提取的质粒作为模版,以设计的PF及PR作为扩增引物进行PCR鉴定。有外源基因RFP-promoter-GFP插入的重组载体,扩增得到的片段长度为1728bp,而无外源插入的pET32a空载体,其扩增产物为946bp。结果(图3)表明,在1700bp处,可见到清晰、特异性的扩增产物条带,所以筛选得到的白斑菌落是含有外源基因的重组载体。

   图3 重组载体pET-mlr promoter的PCR鉴定
结果说明:
1、L1、L2都能扩增得到1700bp的产物,鉴定的重组载体含有外源基因RFP-promoter-GFP
2、M是1000bp Ladder Marker

将经过PCR鉴定的含有重组载体pET-mlr promoter的阳性克隆送到吉林库美生物科技限公司进行测序。结果表明(图4),合成的RFP-promoter-GFP基因序列与设计序列一致

图4 测序结果与设计序列比对

The relationship detection between MLR promoter and microcystin LR

挑取含有重组载体pET-mlr promoter的单菌落,在LB液体培养基中37℃剧烈震荡培养4-5h,待培养液OD600达到0.3-0.4时停止培养,使用灭菌水进行10倍梯度稀释至10-5-10-7,取100ul涂布在含有10ug/L microcystin LR的LB平板上,37℃培养过夜。
将培养的单菌落平板放置在有紫外灯照射的暗室中,观察菌落颜色,可见菌落发出绿色荧光(图5),表明在microcystin LR存在时,mlr promoter可以启动GFP的表达,使E.coli BL21的单菌落在紫外灯下发出绿色荧光。

Fig 5 fluorescence in E. coli colonies viewed under UV light