Team:Jilin China/NOTEBOOK

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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.

Num	Sequence(5' to 3')	Base Count（bp）	Total Synthesis(OD)	Subpackage Pipe Counts	Purification Mode
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
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

The experimental procedure and analysis

1、Dissolving of primers

Sterile deionized water whose volume is determined by primer’s molecular weight is used to dilute the solution of the primer to 10um/L. And it is prepared for the following experiment.

2、Mixing of primers

Oligonucleotides synthesized in powder form need to be centrifuged for 1 min at 10000rpm to assemble powders to the bottom of the EP tube.And be careful to open the cap.
Make sure that the synthesis report is consistent to the OD number on the primer label,and check the number of the dispensing tube before the dissolution of oligonucleotides.The volume of water added to make 10umol/L oligonucleotides’ solution is calculated according to the synthetic single report of each primer. Add sterile deionized water and keep it at room temperature for 2min.And reverse the tube to accelerate the progress of solubilization. Finally, collect substances at the bottom of the tube after centrifugation.
Every six primers are combined to one group.Add 4ulbeginning fiprimer, 1ulintermediate primer and 4ul end primerto a new EP tube, then add sterile deionized water to 20ul, mix them up as spare.

组别	引物编号	吸取量	灭菌去离子水添加量	总体积	每PCR（50ul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

DA-PCR

Every six primers which are single-stranded oligonucleotidesare chemically synthesized were combined to one group. Then they were going to be made as longer double-stranded fragments of intermediate (Block) by DA-PCR.
Procedure of DA-PCR：
Mixed primer solutions                  5μl
Pfu DNA Polymerase 2.5U              0.5μl
dNTP                                4μl
10×Pfu buffer（Mg2+）               5μl
ddH20add to 50μl
Procedure of DA-PCR：
94℃         2min
94℃         30S
50℃         30S    25 cycles
72℃         1min
72℃        10min
4℃preservation
End
DA-PCR splicing reaction product was detected in 2% agarose gel electrophoresis,
DA-PCR product              5μl
10×Loading Buffer         0.6μl   spotting after mixed
100bp Marker              5μl   spotting directly
75V electrophoress for 1h。

4、OE-PCR

    Take the 5 double strand intermediate fragment (Block) spliced by DA-PCR as templates, blocks were further connected by OR-PCR, OE-PCR in a reaction system consisting of:
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
Sterilized ultrapure water   to 50ul
Procedure of OE-PCR：
94℃         2min
94℃         30S
50℃         30S   20 cycles
72℃        2min
72℃       10min
4℃ preservation
End
The full-length gene spliced by OE-PCR was detected by a 1% agarose gel electrophoresis, specific programs are as follows
OE-PCR product           5μl
10×Loading Buffer        0.6μl   spotted after mixing well
100bp Marker              5μl   spotted directly
80V electrophoresis for 1h。

5、Constructing and sequencing of subcloning vector

We design a primer AC-B with Blp I site and a primer AC-S with SgrA I site. Then we use RFP-promoter-GFP as template in PCR. Then we use Blp I and SgrA I enzyme to digest the target fragment and pET32a vector(37℃,3h) and link it overnight(16℃). After this step, transform the linked vector to competent cell(E.coli BL21) and select the desirable colony by using “Blue-White Selection” method.
Constitute of BlpⅠand SgrAⅠdouble enzyme digestion reaction system:
Gene or plasmid                   10μl
BlpⅠ              0.3μl
SgrAⅠ0.3μl
10×NEBuffer 2.12μl
Sterilizing ultrapure watertop up to20μl
Constitute of linking reaction system：
RFP-promoter-GFP                    10μl
pET32a                              3μl
T4 ligase                            0.5μl
10×T4 ligase Buffer                   2μl
Sterilizing ultrapure water           top up to 20μl
Preparation process of competent cell E.coliBL21：
(1) Culturing strain E.coliBL21(37℃)overnight to recovery culture, Adding overnight bacteria culture fluid 600uLto 30 mL LB culture medium, and 37℃，200 r/min shake culturing 1-2 h, until OD600 is around 0.3-0.4.
(2) In the aseptic condition, transfer 30mLbacteria fluid to the centrifugal tube in the ice 10 min to cooling the culture to 0℃.
(3) Centrifuging 10 min in precooling centrifugal machine over 4000r/min, throw supernate, and then add30mLprecooling 0.1mol/L CaCl2 solution to resuspension precipitation in the ice 10 min.
(4) Centrifuging 4000r/min in 4℃10min, throw supernate, each 30mL initial culture resuspension cell precipitation with 1.0mL precooling 0.1mol/LCaCl2 solution, and then 200μL each one.
Heal shock transformation process：
(1) Thawing freshly prepared or -80℃ competentcellBL21suspension.
(2) Add constructed recombinant cloning vector plasmidspSB1C3-A(no more than 50ng, 10μl), shake gently, and then in the ice 30min.
(3) Put EP tube in 42℃ water bath90s，and then transfer it to ice water bath to cool cells1-2 min.
(4) add 800μL LB medium to EP tube，transfer it to 37℃ table，150r/min，to recovery strain 45 min.
Spread plate and Culture
(1)After culture in 37℃, centrifuge the cell solution(5000rpm,5min) and concentrate it to 200μl. Then coat it to LB culture with chloramphenicol and set a positive control.
(2) Wait until the solution in culture is full absorbed by cell, then invert the plate and culture it in 37℃ overnight.
Sequence test
On the pET32a vector(as shown in Fig 1), use the RFP-promoter-GFP gene (1535bp) to replace the fragment between BlpⅠ site and SgrA I site (753bp) and then we get the reconstructed plasmid, pET-mlr promoter.

Fig 3 Map of pET-mlr promoter
Select the white colony on culture and culture it. Then we extract the plasmid and use it as template in PCR to identify the colony. In theory, the reconstructed vector with RFP-promoter-GFP fragment is 1728bp and the empty vector without mlrA fragment is 946bp. So, the result shows that the white colony we select has reconstructed vector with mlrA fragment in it.

Figure 3 PCR identification of recombinant vector pET-mlr promoter

Illustration of the outcome：
1、L1, L2 could be amplified to 1700bp products, the recombinant vector contained a foreign gene RFP-promoter-GFP according to the result of the identification.
2、M was 1000bp Ladder Marker
The positive clones which were proved to contain the recombinant vectors pET-mlr promoter by PCR identification. were sent to 吉林库美生物科技限公司to be sequenced. The results showed (Figure 4), RFP-promoter-GFP sequence is consistent with the design sequence.

Figure 4 Sequencing results with the design sequence alignment

The relationship detection between MLR promoter and microcystin LR

Single colony containingthe recombinant vector pET-mlr promoterwas picked ,vigorously shaken culture for 4-5h in LB liquid medium at 37 ℃，the culturing stopped until OD600 reached 0.3 - 0.4, 10-fold dilution to 10-5-10-7with sterilized water, taking 100ul to coat on the LB plate containing 10ug / L microcystin LR, 37 ℃ for overnight.
Single cultured colony was placed in the UV lamp darkroom, we observed colony color and found that it luminesced green light, indicating that when there is microcystin LR, mlr promoter can promote the expression of GFP, which makes the single colony of E.coli BL21 luminescing green fluorescence in the UV lamp.

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