Team:Jilin China/RESULT

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Latest revision as of 03:59, 18 October 2014

Welcome!
Team Jilin China

NOTEBOOK

Synthesis of gene mlrA

The experimental scheme

1、The amino acid sequence of the protein

MlrA GenBank: AF411068 SOURCE Sphingomonas sp. ACM-3962

MREFVRQRPLLCFYVLAILIALAAHALRAMSPTPLDPMFKMLQETHAHLNIITAVRSTFEYPGAYTLLLFPAAPMFAALIATGIGYGQAGFRELLSRCAPWRSPVSWRQGVTVIAVCFLAFFALTGIMWVQTYLYAPPGTLDRTFLRYGSDPVAIYVMLAASLLLSPGPLLEELGWRGFALPQLLKKFDPLTAAVILGIMWWAWHLPRDLPTLFSGAPGAAWSVIVKQLVITPGFIASTIIAVFVCNKLGGSMWGGVLTHAIHNELGVNVTAEWAPTVAGLGWRPWDLIEFAVAIGLVLICGRSLGAASPDNARLAWGNVPPKLPGGVGDKSGANA

2、The selection of codons

Lactococcus lactis subsp. cremoris SK11 [gbbct]: 2504 CDS's (696252 codons)

http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622

fields: [triplet] [frequency: per thousand] ([number])

UUU 36.1( 25117) UCU 16.5( 11473) UAU 28.4( 19782) UGU 3.9( 2729)

UUC 12.0( 8326) UCC 3.3( 2266) UAC 8.1( 5665) UGC 1.1( 771)

UUA 31.0( 21599) UCA 21.1( 14698) UAA 2.5( 1708) UGA 0.7( 498)

UUG 21.2( 14782) UCG 3.9( 2685) UAG 0.4( 298) UGG 10.1( 7000)

CUU 25.2( 17574) CCU 11.9( 8253) CAU 13.3( 9269) CGU 14.6( 10159)

CUC 8.1( 5614) CCC 2.9( 1990) CAC 4.5( 3121) CGC 4.5( 3122)

CUA 8.0( 5591) CCA 14.5( 10061) CAA 31.1( 21678) CGA 5.9( 4140)

CUG 6.4( 4476) CCG 2.9( 2010) CAG 6.6( 4596) CGG 2.3( 1570)

AUU 51.0( 35505) ACU 20.4( 14218) AAU 40.1( 27903) AGU 14.7( 10220)

AUC 16.1( 11197) ACC 7.4( 5135) AAC 11.1( 7709) AGC 6.2( 4328)

AUA 8.7( 6023) ACA 22.1( 15386) AAA 61.2( 42611) AGA 8.0( 5557)

AUG 24.7( 17203) ACG 7.1( 4919) AAG 13.5( 9375) AGG 1.7( 1152)

GUU 30.8( 21415) GCU 30.1( 20949) GAU 37.3( 25965) GGU 23.4( 16287)

GUC 12.7( 8841) GCC 12.1( 8400) GAC 14.4( 10018) GGC 8.5( 5906)

GUA 12.8( 8921) GCA 22.3( 15521) GAA 56.1( 39035) GGA 24.5( 17046)

GUG 9.2( 6376) GCG 8.2( 5708) GAG 13.0( 9070) GGG 8.2( 5732)

Coding GC 36.75% 1st letter GC 48.61% 2nd letter GC 34.45% 3rd letter GC 27.20%

The codon table used

* TAA

A GCT

C TGT

D GAT

E GAA

F TTT

G GGA

H CAT

I ATT

K AAA

L TTA

M ATG

N AAT

P CCA

Q CAA

R CGA

S TCA

T ACT

V GTT

W TGG

Y TAT

3、The inverse translation sequences and the selection of restriction enzymes

EcoR I

CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT

4、The sequence analysis

_Base Count : 1008 bp (270 A, 377 T, 158 C, 203 G)
_Composition : 36% GC, 64% AT

Absent Sites
AarI AatII Acc65I AccI AciI AclI AcuI AfeI AflII AflIII AgeI AhdI AleI ApaI ApaLI AscI AseI AsiSI AsuII AvaI AvrII BaeGI BamHI BanI BbsI BbvCI BceAI BciVI BclI BfaI BfuAI BglI BglII BlpI BmgBI BmrI BmtI Bpu10I BpuEI BsaAI BsaHI BsaI BsaWI BseMII BseYI BsgI BsiEI BsiWI BslI BsmAI BsmBI BsmFI BsmI BsoBI BspCNI BspEI BspHI BspMI BspQI BsrBI BsrDI BsrFI BsrGI BssHII BstAPI BstBI BstEII BstUI BstZ17I Bsu36I BtgZI BtrI BtsI Cac8I ClaI CviQI DdeI DrdI EaeI EagI EarI EciI Eco31I EcoNI EcoO109I EcoRI EcoRV Esp3I FauI FseI FspAI FspI HaeII HaeIII HgaI HhaI HinP1I HincII HindIII HinfI HpaI HpaII Hpy99I HpyAV HpyCH4IV HpyCH4V KasI KpnI MaeI MaeII MfeI MluI MlyI MscI NaeI NarI NciI NdeI NgoMIV NheI NmeAIII NotI NruI NsiI NspI PacI PasI PciI PflFI PflMI PleI PmeI PpuMI PshAI PsiI PspOMI PspXI PstI PvuI RsaI RsrII SacII SalI SanDI SbfI ScaI SexAI SfaNI SfcI SfiI SfoI SgrAI SmaI SmlI SnaBI SpeI SphI SrfI StuI TaiI TatI TauI TfiI TspGWI TspMI TspRI Tth111I XbaI XhoI XmaI ZraI

Unique Sites

XmnI (6) MslI (25)

Tsp45I (89)

SwaI (142) SspI (147)

MspA1I (211)

DraIII (286)

BstXI (309)

BsaBI (439) Hpy188I (447)

Sau96I (501)

MboII (513)

BssSI (619)

SacI (646) XcmI (651) AlwNI (652)

NlaIV (821)

Hpy166II (901)

5、Sequence Split

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 .1   529bp   1..529
Left  - 5' CCGGAATTCATGCGAGAATTTGTTCG 3'
Rght  - 5' ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAG 3'
RghtU - 5' ATTCTTCTAAUAATGGTCCTGGTGATAATAATAATGAAG 3'
Sequence:

CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAAT

Assembly Oligos: average overlap Tm is 47°;average oligo length is 58bp.

CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAAT
CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCT AGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTT CTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTT GCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTT ACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGT TTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTT CGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCA
ATAATACAAAAATACAAAATCGATAAAATTAACGAAATCGACGAGTACGAAATGCTC AATCTAGGTTACAAATTTTACAATGTTCTTTGAGTACGAGTAAATTTATAATAATGACGA ACTTATAGGTCCTCGAATATGAAATAATAATAAAGGTCGACGAGGTTACAAACGAC AACCTATACCTGTTCGACCTAAAGCTCTTAATAATAGTGCTACACGAGGTACCGCT GTTCCTCAATGACAATAACGACAAACAAAAAATCGAAAAAAACGAAATTGACCTTAATAC ATATACGAGGTGGTCCTTGAAATCTAGCTTGAAAAAATGCTATACCTAGTCTAGGTCAAC TCGACGAAGTAATAATAATAGTGGTCCTGGTAATAATCTTCTTA
GGCCTTAAGTACGCTCTTAAACAAGCTGTTGCTGGTAATAATACAAAAATACAAAATCGATAAAATTAACGAAATCGACGAGTACGAAATGCTCGATACAGTGGTTGAGGTAATCTAGGTTACAAATTTTACAATGTTCTTTGAGTACGAGTAAATTTATAATAATGACGACAAGCTAGTTGAAAACTTATAGGTCCTCGAATATGAAATAATAATAAAGGTCGACGAGGTTACAAACGACGAAATTAACGATGACCTTAACCTATACCTGTTCGACCTAAAGCTCTTAATAATAGTGCTACACGAGGTACCGCTAGTGGTCAAAGTACCGCTGTTCCTCAATGACAATAACGACAAACAAAAAATCGAAAAAAACGAAATTGACCTTAATACACCCAAGTTTGAATAAATATACGAGGTGGTCCTTGAAATCTAGCTTGAAAAAATGCTATACCTAGTCTAGGTCAACGATAAATACAATACAATCGACGAAGTAATAATAATAGTGGTCCTGGTAATAATCTTCTTA

Building Block .2   513bp   519..1031
Left  - 5' ATTAGAAGAATTAGGATGGCGAGGATTTGC 3'
LeftU - 5' ATTAGAAGAAUTAGGATGGCGAGGATTTGC 3'
Rght  - 5' AAGACGTCCTATTAAGCATTAGCTCCTG 3'
Sequence:

ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGGACGTCTT

Assembly Oligos: average overlap Tm is 49°;average oligo length is 58bp.

ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGGACGTCTT
ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCC TGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATT AGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTC TGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGC AGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGA ATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTC TTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGC
TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA
TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA

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.

Num	Tm	MW(g/mole)	nmol/Tube	Add buffer for 10μM/uL
A101	68.3	18451	1.6	155
A102	65	17588.4	1.4	142
A103	69.7	18359	1.5	155
A104	64.2	18533	1.4	143
A105	66.3	18354	1.6	155
A106	68	17321.2	1.5	151
A107	71.1	18533	1.6	156
A108	70.9	17127.2	1.6	164
A109	73.8	18513	1.6	156
A110	66.3	18440	1.4	137
A111	67.6	18426	1.6	156
A112	69.7	18506	1.5	147
A113	67	18441	1.6	156
A114	64	13568.8	2.0	200
A201	66.3	18568	1.4	143
A202	64.9	18407	1.5	145
A203	68.3	18522	1.5	155
A204	71.8	16968	1.6	162
A205	68.3	18491	1.5	154
A206	64.9	18492	1.4	138
A207	67.9	17753.4	1.6	156
A208	68.3	18305	1.5	151
A209	72.6	17117	1.6	164
A210	70.4	18278	1.5	149
A211	68.6	17622.4	1.7	165
A212	72.6	18145.8	1.6	156
A213	71.9	18356.8	1.5	146
A214	66.9	13072.6	2.2	220
A1	62	8002.2	3.6	356
A2	61	12043.8	2.2	222
A3	62	9375	2.8	281
A4	62	8548.6	3.3	330
A5	62	8548.6	3.3	330

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.

Group	Number of primer	Uptake	The volume of sterile deionized water	The total volume	Each PCR (50ul system) uptake	Final concentration
A1	A101	4	8	20	5ul	200nM
	A102	1				50nM
	A103	1				50nM
	A104	1				50nM
	A105	1				50nM
	A106	4				200nM
A2	A107	4	8	20	5ul	200nM
	A108	1				50nM
	A109	1				50nM
	A110	1				50nM
	A111	1				50nM
	A112	4				200nM
A3	A113	4	8	20	5ul	200nM
	A114	1				50nM
	A201	1				50nM
	A202	1				50nM
	A203	1				50nM
	A204	4				200nM
A4	A205	4	8	20	5ul	200nM
	A206	1				50nM
	A207	1				50nM
	A208	1				50nM
	A209	1				50nM
	A210	4				200nM
A5	A211	4	10	20	5ul	200nM
	A212	1				50nM
	A213	1				50nM
	A214	4				200nM

3、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。

Figure1 DA-PCR splicing results

Illustration of the outcome:
1、A1, A2, A3, A4 could getintermediates (Block1-4) which were made by six single-stranded oligonucleotides splicedtogether, but the A3 and A4 groups could also see by-products which were made by four single-stranded oligonucleotides splicedtogether , and to be made a whole one after recycling of Agarose gel.
2、A5 could obtainintermediates(Block5) which were made by four single-stranded oligonucleotides splicedtogether.

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
A1                         1μl
A4                        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
47℃         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.

Illustration of the outcome:

L1, L2, L3 could get mlrA genome which were made by five single-stranded oligonucleotides splicedtogether.
Wide strip of the tape at 100bp was caused by excessive amount of amplification primers A1 and A4.

5、Constructing and sequencing of subcoloning vector

After double enzyme digestion reaction at 37℃ for 3h by EcoRⅠand PstⅠ, MlrA gene and pSB1C3 vector linked together at 16℃ overnight. And then transformed to E.coli JM109 competent cell, and select the desirable colony by using “blue-white selection” method.
Constitute of EcoRⅠand PstⅠdouble enzyme digestion reaction system：
Gene or plasmid                      10μl
EcoRⅠ                             0.4μl
PstⅠ                               0.2μl
10×NEBuffer 3.1                     2μl
sterilizing ulturapure water        top up to 20μl
Constitute of linking reaction system：
mlrA Gene                           10μl
pSB1C3                              3μl
T4 ligase                            0.5μl
10×T4 ligase Buffer                   2μl
sterilizing ulturapure 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
Replace the fragment(45bp) between EcoR I site and Pst I site with mlrA(1024bp) on pSB1C3 vector and then we get the reconstructed plasmid, pSB1C3-A.

Fig3.Reconstructed vector pSB1C3-A

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 mlrA fragment is 1293bp and the empty vector without mlrA fragment is 314bp. So, the result shows that the white colony we select has reconstructed vector with mlrA fragment in it.

Fig 4. The identification of reconstructed vector pSB1C3-A

Results:

1.L1 and L2 is 1300bp and it confirms that the reconstructed vector has mlrA on it.

2.M is the 100bp Ladde Marker.

Sequencing result shows that the sequence of synthetic mlrA gene is exactly same as sequence of designed sequence.

Fig5.Comparison of result of sequencing and designed sequence

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+<!--  here ends the section that changes the default wiki template to a white full width background -->
-<div style="width:965px;display:block;word-break: break-all;word-wrap: break-word;">
-<h1 >mlrA基因合成 </h1>
-<h2 >一、实验方案 </h2>
+<!-- beginning of your page -->
-<h3 >1、目的蛋白氨基酸序列 </h3>
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+<!-- You can replace this image with your team's logo! -->
+<h3>Welcome!  <br>Team Jilin China</h3>
+</td>
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+<!-- end of first block -->
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+<div class="observation"><table width="100%"  cellspacing="0" height="0px">
+<tr><td  bgColor="#FEE5AD"></td>
+<td width="975px" align="left" bgColor="#FEE5AD" >
+<table  width="975px"  cellspacing="0" height="0px">
+<tr>
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+<!-- clear the floats if required -->
+<div class="clear"> </div>
+<meta http-equiv="Content-Type" content="text/html; charset=gb2312" />
+<title>NOTEBOOK</title>
+</head>
+<!--内容-->
+<div id="coop">
+<center><h2>Synthesis of gene mlrA</h2></center>
+<h3>The experimental scheme</h3>
+<h3>1、The amino acid sequence of the protein</h3>
 <p >MlrA&nbsp;GenBank:&nbsp;AF411068&#9;SOURCE&nbsp;&nbsp;Sphingomonas&nbsp;sp.&nbsp;ACM-3962 </p>
 <p >MREFVRQRPLLCFYVLAILIALAAHALRAMSPTPLDPMFKMLQETHAHLNIITAVRSTFEYPGAYTLLLFPAAPMFAALIATGIGYGQAGFRELLSRCAPWRSPVSWRQGVTVIAVCFLAFFALTGIMWVQTYLYAPPGTLDRTFLRYGSDPVAIYVMLAASLLLSPGPLLEELGWRGFALPQLLKKFDPLTAAVILGIMWWAWHLPRDLPTLFSGAPGAAWSVIVKQLVITPGFIASTIIAVFVCNKLGGSMWGGVLTHAIHNELGVNVTAEWAPTVAGLGWRPWDLIEFAVAIGLVLICGRSLGAASPDNARLAWGNVPPKLPGGVGDKSGANA </p>
-<h3 >2、密码子选择 </h3>
+<h3 >2、The selection of codons</h3>
-<p >Lactococcus&nbsp;lactis&nbsp;subsp.&#160;cremoris&nbsp;SK11&#160;[gbbct]:&nbsp;2504&nbsp;CDS's&nbsp;(696252&nbsp;codons) </p>
+<h4>Lactococcus&nbsp;lactis&nbsp;subsp.&#160;cremoris&nbsp;SK11&#160;[gbbct]:&nbsp;2504&nbsp;CDS's&nbsp;(696252&nbsp;codons) </h4>
-<p >http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622 </p>
+<p ><a href="http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622">http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272622</a> </p>
-<p ><img width="634" height="1" src="../AppData/Roaming/Adobe/Dreamweaver CC/zh_CN/OfficeImageTemp/wps2B49.tmp.png" ></p>
 <p >fields:&nbsp;[triplet]&nbsp;[frequency:&#160;per&nbsp;thousand]&nbsp;([number]) </p>
-<p ><img width="634" height="1" src="../AppData/Roaming/Adobe/Dreamweaver CC/zh_CN/OfficeImageTemp/wps2B4A.tmp.png" ></p>
 <p >UUU&nbsp;36.1(&nbsp;25117)&nbsp;&nbsp;UCU&nbsp;16.5(&nbsp;11473)&nbsp;&nbsp;UAU&nbsp;28.4(&nbsp;19782)&nbsp;&nbsp;UGU&nbsp;&nbsp;3.9(&nbsp;&nbsp;2729) </p>
 <p >UUC&nbsp;12.0(&nbsp;&nbsp;8326)&nbsp;&nbsp;UCC&nbsp;&nbsp;3.3(&nbsp;&nbsp;2266)&nbsp;&nbsp;UAC&nbsp;&nbsp;8.1(&nbsp;&nbsp;5665)&nbsp;&nbsp;UGC&nbsp;&nbsp;1.1(&nbsp;&nbsp;&nbsp;771) </p>
@@ Line 38: / Line 239: @@
 <p >GUA&nbsp;12.8(&nbsp;&nbsp;8921)&nbsp;&nbsp;GCA&nbsp;22.3(&nbsp;15521)&nbsp;&nbsp;GAA&nbsp;56.1(&nbsp;39035)&nbsp;&nbsp;GGA&nbsp;24.5(&nbsp;17046) </p>
 <p >GUG&nbsp;&nbsp;9.2(&nbsp;&nbsp;6376)&nbsp;&nbsp;GCG&nbsp;&nbsp;8.2(&nbsp;&nbsp;5708)&nbsp;&nbsp;GAG&nbsp;13.0(&nbsp;&nbsp;9070)&nbsp;&nbsp;GGG&nbsp;&nbsp;8.2(&nbsp;&nbsp;5732) </p>
-<p ><img width="634" height="1" src="../AppData/Roaming/Adobe/Dreamweaver CC/zh_CN/OfficeImageTemp/wps2B5A.tmp.png" ></p>
 <p >Coding&nbsp;GC&nbsp;36.75%&nbsp;1st&nbsp;letter&nbsp;GC&nbsp;48.61%&nbsp;2nd&nbsp;letter&nbsp;GC&nbsp;34.45%&nbsp;3rd&nbsp;letter&nbsp;GC&nbsp;27.20% </p>
-<p ><img width="634" height="1" src="../AppData/Roaming/Adobe/Dreamweaver CC/zh_CN/OfficeImageTemp/wps2B5B.tmp.png" >密码子密码子密码子 </p>
+<h4>The codon table used</h4>
-<p >&nbsp;</p>
-<p >使用密码子表 </p>
 <p >*&nbsp;TAA&nbsp; </p>
 <p >A&nbsp;GCT </p>
@@ Line 64: / Line 262: @@
 <p >W&nbsp;TGG </p>
 <p >Y&nbsp;TAT </p>
-<p >&nbsp;</p>
+<h3>3、The inverse translation sequences and the selection of restriction enzymes</h3>
-<h3 >3、逆翻译序列及限制性内切酶选择 </h3>
 <p >EcoR&nbsp;I </p>
 <p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCTATTTTAATTGCTTTAGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTTACAAGAAACTCATGCTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTTTATTATTATTTCCAGCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTTCGAGAATTATTATCACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGTTTGTTTTTTAGCTTTTTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTTAGATCGAACTTTTTTACGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCACCAGGACCATTATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCCATTAACTGCTGCTGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATTTTCAGGAGCTCCAGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTCAACTATTATTGCTGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGCTATTCATAATGAATTAGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGACCATGGGATTTAATTGAATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTCACCAGATAATGCTCGATTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGCTAATGCTTAATAGCTGCAGTT </p>
+<h3 >4、The sequence analysis</h3>
-<h3 >4、序列分析 </h3>
 <p >_Base&nbsp;Count&nbsp;:&nbsp;1008&nbsp;bp&nbsp;(270&nbsp;A,&nbsp;377&nbsp;T,&nbsp;158&nbsp;C,&nbsp;203&nbsp;G)<br>
    &#160;_Composition&nbsp;:&nbsp;36%&nbsp;GC,&nbsp;64%&nbsp;AT<br>
@@ Line 91: / Line 286: @@
 <p >NlaIV&nbsp;(821) </p>
 <p >Hpy166II&nbsp;(901) </p>
-<h3 >5、序列拆分 </h3>
+<h3 >5、Sequence Split</h3>
 <p >The&nbsp;maximum&nbsp;allowable&nbsp;assembly&nbsp;oligo&nbsp;length&nbsp;is&nbsp;equal&nbsp;to&nbsp;the&nbsp;target&nbsp;assembly&nbsp;oligo&nbsp;length&nbsp;(60).&nbsp;This&nbsp;may&nbsp;cause&nbsp;some&nbsp;weird&nbsp;behavior,&nbsp;especially&nbsp;in&nbsp;terms&nbsp;of&nbsp;overlap&nbsp;melting&nbsp;temperature. </p>
 <p >&nbsp;</p>
@@ Line 120: / Line 315: @@
    &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;TGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCAC&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;TGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAA&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;ATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCC&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;CCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTAC&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;ACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCA&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;TGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTG&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;CTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA<br>
    TAATCTTCTTAATCCTACCGCTCCTAAACGAAATGGTGTTAATAATTTTTTTAAACTAGGTAATTGACGACGACAATAAAATCCTTAATACACCACCCGAACCGTAAATGGTGCTCTAAATGGTTGAAATAAAAGTCCTCGAGGTCCTCGACGAACCAGTCAATAACAATTTGTTAATCAATAATGAGGTCCTAAATAACGAAGTTGATAATAACGACAAAAACAAACATTATTTAATCCTCCTAGTTACACCCCTCCTCAAAATTGAGTACGATAAGTATTACTTAATCCTCAATTACAATGACGACTTACCCGAGGTTGACAACGACCTAATCCTACCGCTGGTACCCTAAATTAACTTAAACGACAACGATAACCTAATCAAAATTAAACACCTGCTAGTAATCCTCGACGAAGTGGTCTATTACGAGCTAATCGAACCCCTTTACAAGGTGGTTTTAATGGTCCTCCTCAACCTCTATTTAGTCCTCGATTACGAATTATCCTGCAGAA </p>
+<h3>The experimental procedure and analysis</h3>
+<h3 >1、Dissolving of primers</h3>
-<h2 >二、实验操作及结果分析 </h2>
+<p >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. </p>
-<h3 >1、引物溶解 </h3>
-<p >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。 </p>
 <table align="center" >
    <tr >
-     <td width="47" valign="center" ><p >引物编号 </p></td>
+     <td width="47" valign="center" ><p >Num</p></td>
      <td width="48" valign="center" ><p >Tm </p></td>
      <td width="85" valign="center" ><p >MW(g/mole) </p></td>
@@ Line 365: / Line 558: @@
    </tr>
 </table>
-<p >&nbsp;</p>
+<h3 id="coop">2、Mixing of primers</h3>
-<h3 >2、引物混合 </h3>
+<p id="coop">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. </p>
-<p >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。 </p>
+<p id="coop">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.</p>
-<p >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。 </p>
+<p id="coop">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.</p>
-<p >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。 </p>
+<table align="center" border="1" cellspacing="0" cellpadding="0" width="612">
-<table align="center" >
+   <tr>
-   <tr >
+     <td width="67"><p align="center"><strong>Group</strong></p></td>
-     <td width="67" valign="center" ><p >组别 </p></td>
+     <td width="103"><p align="center"><strong>Number of primer</strong></p></td>
-     <td width="103" valign="center" ><p >引物编号 </p></td>
+     <td width="65"><p align="center"><strong>Uptake</strong></p></td>
-     <td width="65" valign="center" ><p >吸取量 </p></td>
+     <td width="85"><p align="center"><strong>The volume of sterile deionized water</strong></p></td>
-     <td width="85" valign="center" ><p >灭菌去离子水添加量 </p></td>
+     <td width="75"><p align="center"><strong>The total volume</strong></p></td>
-     <td width="74" valign="center" ><p >总体积 </p></td>
+     <td width="108" valign="top"><p align="center"><strong>Each PCR (50ul system) uptake</strong></p></td>
-     <td width="108" valign="top" ><p >每PCR（50ul体系）吸取量 </p></td>
+     <td width="108"><p align="center"><strong>Final concentration</strong></p></td>
-     <td width="108" valign="center" ><p >终浓度 </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="67" valign="center" rowspan="6" ><p >A1 </p></td>
+     <td width="67" rowspan="6"><p align="center">A1</p></td>
-     <td width="103" valign="center" ><p >A101 </p></td>
+     <td width="103"><p align="left">A101</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
+     <td width="85" rowspan="6"><p align="center">8</p></td>
-     <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
+     <td width="75" rowspan="6"><p align="center"><a name="_GoBack"></a>20</p></td>
-     <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
+     <td width="108" rowspan="6"><p align="center">5ul</p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A102 </p></td>
+     <td width="103"><p align="left">A102</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="center" ><p >50nM </p></td>
+     <td width="108"><p align="center">50nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A103 </p></td>
+     <td width="103"><p align="left">A103</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A104 </p></td>
+     <td width="103"><p align="left">A104</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A105 </p></td>
+     <td width="103"><p align="left">A105</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A106 </p></td>
+     <td width="103"><p align="left">A106</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="67" valign="center" rowspan="6" ><p >A2 </p></td>
+     <td width="67" rowspan="6"><p align="center">A2</p></td>
-     <td width="103" valign="center" ><p >A107 </p></td>
+     <td width="103"><p align="left">A107</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
+     <td width="85" rowspan="6"><p align="center">8</p></td>
-     <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
+     <td width="75" rowspan="6"><p align="center">20</p></td>
-     <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
+     <td width="108" rowspan="6"><p align="center">5ul </p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A108 </p></td>
+     <td width="103"><p align="left">A108</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="center" ><p >50nM </p></td>
+     <td width="108"><p align="center">50nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A109 </p></td>
+     <td width="103"><p align="left">A109</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A110 </p></td>
+     <td width="103"><p align="left">A110</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A111 </p></td>
+     <td width="103"><p align="left">A111</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A112 </p></td>
+     <td width="103"><p align="left">A112</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="67" valign="center" rowspan="6" ><p >A3 </p></td>
+     <td width="67" rowspan="6"><p align="center">A3</p></td>
-     <td width="103" valign="center" ><p >A113 </p></td>
+     <td width="103"><p align="left">A113</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
+     <td width="85" rowspan="6"><p align="center">8</p></td>
-     <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
+     <td width="75" rowspan="6"><p align="center">20</p></td>
-     <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
+     <td width="108" rowspan="6"><p align="center">5ul </p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A114 </p></td>
+     <td width="103"><p align="left">A114</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="center" ><p >50nM </p></td>
+     <td width="108"><p align="center">50nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A201 </p></td>
+     <td width="103"><p align="left">A201</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A202 </p></td>
+     <td width="103"><p align="left">A202</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A203 </p></td>
+     <td width="103"><p align="left">A203</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A204 </p></td>
+     <td width="103"><p align="left">A204</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="67" valign="center" rowspan="6" ><p >A4 </p></td>
+     <td width="67" rowspan="6"><p align="center">A4</p></td>
-     <td width="103" valign="center" ><p >A205 </p></td>
+     <td width="103"><p align="left">A205</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="85" valign="center" rowspan="6" ><p >8 </p></td>
+     <td width="85" rowspan="6"><p align="center">8</p></td>
-     <td width="74" valign="center" rowspan="6" ><p >20 </p></td>
+     <td width="75" rowspan="6"><p align="center">20</p></td>
-     <td width="108" valign="center" rowspan="6" ><p >5ul </p></td>
+     <td width="108" rowspan="6"><p align="center">5ul </p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A206 </p></td>
+     <td width="103"><p align="left">A206</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="center" ><p >50nM </p></td>
+     <td width="108"><p align="center">50nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A207 </p></td>
+     <td width="103"><p align="left">A207</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A208 </p></td>
+     <td width="103"><p align="left">A208</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A209 </p></td>
+     <td width="103"><p align="left">A209</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A210 </p></td>
+     <td width="103"><p align="left">A210</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="67" valign="center" rowspan="4" ><p >A5 </p></td>
+     <td width="67" rowspan="4"><p align="center">A5</p></td>
-     <td width="103" valign="center" ><p >A211 </p></td>
+     <td width="103"><p align="left">A211</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="85" valign="center" rowspan="4" ><p >10 </p></td>
+     <td width="85" rowspan="4"><p align="center">10</p></td>
-     <td width="74" valign="center" rowspan="4" ><p >20 </p></td>
+     <td width="75" rowspan="4"><p align="center">20</p></td>
-     <td width="108" valign="center" rowspan="4" ><p >5ul </p></td>
+     <td width="108" rowspan="4"><p align="center">5ul </p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A212 </p></td>
+     <td width="103"><p align="left">A212</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="center" ><p >50nM </p></td>
+     <td width="108"><p align="center">50nM</p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A213 </p></td>
+     <td width="103"><p align="left">A213</p></td>
-     <td width="65" valign="center" ><p >1 </p></td>
+     <td width="65"><p align="center">1</p></td>
-     <td width="108" valign="top" ><p >50nM </p></td>
+     <td width="108" valign="top"><p align="center">50nM </p></td>
    </tr>
-   <tr >
+   <tr>
-     <td width="103" valign="center" ><p >A214 </p></td>
+     <td width="103"><p align="left">A214</p></td>
-     <td width="65" valign="center" ><p >4 </p></td>
+     <td width="65"><p align="center">4</p></td>
-     <td width="108" valign="center" ><p >200nM </p></td>
+     <td width="108"><p align="center">200nM</p></td>
    </tr>
 </table>
-<h3 >3、DA-PCR </h3>
+<h3 id="coop">3、DA-PCR </h3>
-<p >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： </p>
+<p id="coop">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 </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Mixed&nbsp;primer&nbsp;solutions&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l </p>
+<p id="coop">Procedure of DA-PCR： <br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pfu&nbsp;DNA&nbsp;Polymerase&nbsp;2.5U&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.5&#956;l&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </p>
+  Mixed primer solutions                   5μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;dNTP&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4&#956;l </p>
+  Pfu DNA Polymerase  2.5U              0.5μl     <br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10&#215;Pfu&nbsp;buffer（Mg2+）&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l </p>
+  dNTP                                4μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;灭菌超纯水&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;补齐至&nbsp;&nbsp;50&#956;l </p>
+×Pfu buffer（Mg2+）               5μl<br>
-<p >DA-PCR的反应程序为： </p>
+  ddH20add to 50μl<br>
+  Procedure of DA-PCR： <br>
+℃         2min<br>
+℃         30S<br>
+℃         30S     25 cycles<br>
+℃         1min<br>
+℃        10min<br>
+℃         preservation<br>
+  End<br>
+  DA-PCR splicing reaction product was  detected in 2% agarose gel electrophoresis,<br>
+  DA-PCR product              5μl <br>
+×Loading Buffer         0.6μl    spotting after mixed<br>
+bp Marker              5μl    spotting directly<br>
+V electrophoress for 1h。 </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;94&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2min </p>
+<div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/8/84/Simonsong-result-1.png" ></p></div>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;94&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30S </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;47&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30S&nbsp;&nbsp;&nbsp;&nbsp;25个循环 </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;72&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1min </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;72&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10min </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;保存 </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;End </p>
-<p >DA-PCR拼接反应产物检测采用2％的琼脂糖凝胶电泳，其中 </p>
-<p >DA-PCR产物&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l&nbsp; </p>
-<p >10&#215;Loading&nbsp;Buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.6&#956;l&nbsp;&nbsp;&nbsp;混合均匀后点样 </p>
-<p >100bp&nbsp;Marker&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l&nbsp;&nbsp;&nbsp;&nbsp;直接点样 </p>
-<p >75V电泳1h。 </p>
-<p >&#9; </p>
+<p id="coop" align="center">Figure1 DA-PCR  splicing results <br></P>
+ <p id="coop"> Illustration of  the outcome:<br>
+、A1,  A2, A3, A4 could getintermediates (Block1-4) which were made by six  single-stranded oligonucleotides splicedtogether, but the A3 and A4 groups  could also see by-products which were made by four single-stranded  oligonucleotides splicedtogether , and to be made a whole one after recycling  of Agarose gel.<br>
+、A5 could  obtainintermediates(Block5) which were made by four single-stranded  oligonucleotides splicedtogether.</p>
+<h3 id="coop">4、OE-PCR </h3>
+<p id="coop">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: </p>
+<p id="coop">Block1                       1μl<br>
+  Block2                       1μl<br>
+  Block3                       1μl<br>
+  Block4                       1μl<br>
+  Block5                       1μl<br>
+  A1                         1μl<br>
+  A4                        1μl<br>
+  <em>Pfu</em> DNA Polymerase 2.5U       0.5μl <br>
+  dNTP                        4μl<br>
+×<em>Pfu</em> buffer（Mg2+）         5μl<br>
-<p ><img src="https://static.igem.org/mediawiki/2014/8/84/Simonsong-result-1.png" ></p>
+ Sterilized  ultrapure water   to 50ul<br>
+  Procedure of OE-PCR： <br>
+℃         2min<br>
+℃         30S<br>
+℃         30S    20 cycles<br>
+℃        2min<br>
+℃       10min<br>
+℃        preservation<br>
+  End<br>
+  The full-length gene  spliced by OE-PCR was detected by a 1% agarose gel electrophoresis, specific  programs are as follows<br>
+  OE-PCR  product           5μl <br>
+×Loading Buffer        0.6μl    spotted after mixing well<br>
+bp  Marker              5μl   spotted directly<br>
+V electrophoresis for  1h.</p>
+<div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/2/24/Simonsong-result-2.png" ></p>
+</div>
-<p >图1&nbsp;DA-PCR拼接结果 </p>
+<p id="coop">Illustration of  the outcome:</p>
-<p >结果说明： </p>
+<ol id="coop">
-<p >1、A1、A2、A3、A4都能得到6条单链寡核苷酸拼接到一起的中间产物（Block1-4），但是A3和A4组还可看到4条单链寡核苷酸拼接到一起的副产物，需胶回收后再进一步拼全基因。 </p>
+  <li>L1,  L2, L3 could get mlrA genome which were made by five single-stranded  oligonucleotides splicedtogether.</li>
-<p >2、A5组可得到4条单链寡核苷酸拼接到一起的中间产物（Block5）。 </p>
+  <li>Wide  strip of the tape at 100bp was caused by excessive amount of amplification  primers A1 and A4.</li>
-<p >&nbsp;</p>
+</ol>
-<h3 >4、OE-PCR </h3>
+<h3 id="coop">5、Constructing and sequencing of subcoloning vector</h3>
-<p >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： </p>
+<p id="coop">After double  enzyme digestion reaction at 37℃ for 3h by<em> Eco</em>RⅠand <em>Pst</em>Ⅰ, MlrA gene and pSB1C3 vector linked  together at 16℃  overnight. And then transformed to<em> E.coli</em> JM109 competent cell, and select the desirable colony by using &ldquo;blue-white  selection&rdquo; method. <br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Block1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+  Constitute of <strong><em>Eco</em>R</strong><strong>Ⅰ</strong><strong>and <em>Pst</em></strong><strong>Ⅰdouble enzyme  digestion reaction system</strong><strong>：</strong><strong> </strong><br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Block2&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+  Gene or plasmid                       10μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Block3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+  <em>Eco</em>RⅠ                             0.4μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Block4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+  <em>Pst</em>Ⅰ                               0.2μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Block5&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+×NEBuffer 3.1                      2μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;A1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+  sterilizing ulturapure water         top up to 20μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;A4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;1&#956;l </p>
+  <strong>Constitute  of linking reaction system</strong><strong>：</strong><strong> </strong><br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pfu&nbsp;DNA&nbsp;Polymerase&nbsp;2.5U&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.5&#956;l&nbsp; </p>
+  mlrA Gene                           10μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;dNTP&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4&#956;l </p>
+  pSB1C3                               3μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10&#215;Pfu&nbsp;buffer（Mg2+）&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l </p>
+  T4 ligase                             0.5μl<br>
-<p ><img width="349" height="2" src="3_wpsE664.tmp.png" >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;灭菌超纯水&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;补齐至&nbsp;&nbsp;50&#956;l </p>
+×T4 ligase Buffer                    2μl<br>
-<p >OE-PCR反应程序为： </p>
+  sterilizing ulturapure water         top up to 20μl<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;94&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2min </p>
+  Preparation process of competent cell <strong><em>E.coli</em>BL21</strong><strong>：</strong><strong> </strong><br>
-<p ><img width="13" height="64" src="3_wpsE675.tmp.png" >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;94&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30S </p>
+  (1) Culturing strain <em>E.coli</em>BL21(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.<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;47&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;30S&nbsp;&nbsp;&nbsp;&nbsp;20个循环 </p>
+  (2) In the aseptic  condition, transfer 30mLbacteria fluid to the centrifugal tube in the ice 10  min to cooling the culture to 0℃.<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;72&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2min </p>
+  (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.<br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;72&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10min </p>
+  (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.<br>
-<p ><img width="349" height="2" src="3_wpsE686.tmp.png" >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;4&#8451;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;保存 </p>
+  <strong>Heal  shock transformation process</strong><strong>：</strong><strong> </strong><br>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;End </p>
+  (1) Thawing freshly  prepared or -80℃ competentcellBL21suspension.<br>
-<p >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 </p>
+  (2) Add constructed  recombinant cloning vector plasmidspSB1C3-A(no more than 50ng, 10μl), shake  gently, and then in the ice 30min.<br>
-<p ><img width="13" height="34" src="3_wpsE687.tmp.png" >OE-PCR产物&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l&nbsp; </p>
+  (3)  Put EP tube in 42℃ water bath90s，and  then transfer it to ice water bath to cool cells1-2 min.<br>
-<p >10&#215;Loading&nbsp;Buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.6&#956;l&nbsp;&nbsp;&nbsp;混合均匀后点样 </p>
+  (4) add 800μL LB medium  to EP tube，transfer  it to 37℃  table，150r/min，to  recovery strain 45 min.<br>
-<p ><img width="349" height="2" src="3_wpsE688.tmp.png" >100bp&nbsp;Marker&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5&#956;l&nbsp;&nbsp;&nbsp;&nbsp;直接点样 </p>
+  Spread plate and  Culture<br>
-<p >80V电泳1h。 </p>
+  (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.<br>
-<p >&nbsp;</p>
+  (2) Wait until the  solution in culture is full absorbed by cell, then invert the plate and culture  it in 37℃  overnight.<br>
-<p ><img width="265" height="236" src="3_wpsE6A8.tmp.png" ></p>
+  Sequence test<br>
+  Replace the fragment(45bp) between EcoR I site  and Pst I site with mlrA(1024bp) on pSB1C3 vector and then we get the  reconstructed plasmid, pSB1C3-A.</p>
+<div style="text-align:center"><p  id="coop"><img  src="https://static.igem.org/mediawiki/2014/0/0d/Simonsong-result-3.png" >&nbsp;</p></div>
-<p >图2&nbsp;OE-PCR拼接结果 </p>
+<p  id="coop" align="center">Fig3.Reconstructed vector pSB1C3-A </p>
+<p  id="coop">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 mlrA fragment is 1293bp and the empty vector without mlrA fragment is 314bp. So, the result shows that the white colony we select has reconstructed vector with mlrA fragment in it.</p>
+<div style="text-align:center"><p  id="coop"><img  src="https://static.igem.org/mediawiki/2014/6/63/Simonsong-result-4.png" ></p></div>
+<p  id="coop" align="center">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Fig 4. The identification of reconstructed vector pSB1C3-A </p>
+<p  id="coop">Results: </p>
+<p  id="coop">1.L1 and L2 is 1300bp and it confirms that the reconstructed vector has mlrA on it. </p>
+<p  id="coop">2.M is the 100bp Ladde Marker. </p>
+<p  id="coop">Sequencing result shows that the sequence of synthetic mlrA gene is exactly same as sequence of designed sequence. </p>
-<p >结果说明： </p>
-<p >1、L1、L2、L3都能得到5条中间产物拼接到一起的mlrA全基因。 </p>
-<p >2、100bp处的宽条带是由于扩增引物A1和A4加入量过多所导致。 </p>
+<div style="text-align:center"><p  id="coop"><img src="https://static.igem.org/mediawiki/2014/5/5a/Simonsong-result-5.png" ><img src="https://static.igem.org/mediawiki/2014/0/0b/Simonsong-result-6.png" >&nbsp;</p></div>
+<p  id="coop"align="center" > Fig5.Comparison of result of sequencing and designed sequence </p>
+<p  id="coop">&nbsp;</p>
-<h3 >5、亚克隆载体构建与测序 </h3>
-<p >合成的mlrA基因与pSB1C3载体经过EcoR&#8544;和Pst&#8544;于37&#8451;双酶切3h后，16&#8451;连接过夜，转化到E.coli&nbsp;JM109感受态细胞中，经蓝白筛选挑取白色菌落。 </p>
-<p >EcoR&#8544;和Pst&#8544;双酶切反应体系组成为： </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Gene&nbsp;or&nbsp;plasmid&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;EcoR&#8544;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.4&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Pst&#8544;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.2&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10&#215;NEBuffer&nbsp;3.1&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;灭菌超纯水&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;补齐至&nbsp;&nbsp;20&#956;l </p>
-<p >连接反应体系组成为： </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;mlrA&nbsp;Gene&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;pSB1C3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;T4&nbsp;ligase&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;0.5&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;10&#215;T4&nbsp;ligase&nbsp;Buffer&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;2&#956;l </p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;灭菌超纯水&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;补齐至&nbsp;&nbsp;20&#956;l </p>
-<p >E.coli&nbsp;JM109感受态细胞制备流程： </p>
-<p >(1)&nbsp;E.coli&nbsp;JM109菌株37&#8451;过夜培养以复苏菌种，取过夜培养的菌液600uL加入到30&nbsp;mL&nbsp;LB培养基中，37&#8451;，200&nbsp;r/min振荡培养1-2&nbsp;h，至OD600约为0.3-0.4左右。 </p>
-<p >(2)&nbsp;无菌条件下，将30mL菌液转移至离心管中，冰上放置10&nbsp;min，使培养物冷却至0&#8451;。 </p>
-<p >(3)&nbsp;在预冷4&#8451;的离心机上以4000r/min离心10min，弃去上清，加入30mL预冷的0.1mol/L&nbsp;CaCl2溶液重悬沉淀，冰浴上放置10&nbsp;min。 </p>
-<p >(4)&nbsp;以4000r/min在4&#8451;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，200&#956;L/管分装。 </p>
-<p >热激转化过程： </p>
-<p >(1)&nbsp;取新鲜制备或者从-80&#8451;冰箱中取出感受态细胞JM109悬液，冰浴解冻。 </p>
-<p >(2)&nbsp;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10&#956;l)，轻轻摇匀，冰上放置30分钟后。 </p>
-<p >(3)&nbsp;将该EP管放入预加温至42&#8451;的水浴中，放置90s，快速转移到冰浴中，使细胞冷却1-2&nbsp;min。 </p>
-<p >(4)&nbsp;向EP管加入800&nbsp;&#956;L&nbsp;LB培养基，转移至37&#8451;摇床上，150r/min，温育45&nbsp;min以复苏菌株。&nbsp; </p>
-<p >平板涂布及培养过程： </p>
-<p >(1)&nbsp;将37&#8451;培养后的菌体低速离心（5000rpm、5min）浓缩为200&#956;l，涂布到含有氯霉素的LB平板上，设阳性对照。 </p>
-<p >(2)&nbsp;将平板置于室温下至液体被完全吸收，倒置平皿，37&#8451;过夜培养； </p>
-<p >实验连接组：连接产物，氯霉素板 </p>
-<p >阳性对照组:&nbsp;氯霉素板，以pSB1C3质粒代替DNA溶液,&nbsp;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 </p>
-<p >阴性对照组：氯霉素板，为连接时用水代替酶，其它与实验组相同。 </p>
-<p >空菌组：感受态细胞，无氯霉素板 </p>
-<p >序列测定 </p>
-<p >在pSB1C3载体上，将EcoR&nbsp;I和Pst&nbsp;I之间的45bp片段使用mlrA的1024bp的片段替换，将得到重组的pSB1C3-A质粒（图3）。 </p>
+<div style="background:#FEE5AD;">
+<a height="30px" width="5%" align="center" onMouseOver="this.bgColor='#FFFFFF'" onMouseOut="this.bgColor='#A1DBB2'"  href="#menu" style="text-decoration:none;color:#1C140D;float:right;background:#A1DBB2;"> Top </a>
+</div>
+</div>
+<td width="100%" rowspan="3" align="left">
+<td> </td>
+<!-- Content goes here -->
+<!-- end of Block-->
-<p ><img width="624" height="585" src="5_wps5227.tmp.png" >&nbsp;</p>
-<p >图3&nbsp;重组载体pSB1C3-A </p>
-<p >挑取白色菌落，经液体培养后，提取的质粒作为模版，以VF2及VR作为扩增引物进行PCR鉴定。有外源基因mlrA插入的重组载体，扩增得到的片段长度为1293bp，而无外源插入的pSB1C3空载体，其扩增产物为314bp。结果（图4）表明，在1300bp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。 </p>
-<p ><img width="389" height="370" src="5_wps5238.tmp.png" ></p>
-<p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;图4&nbsp;重组载体pSB1C3-A的PCR鉴定 </p>
-<p >结果说明： </p>
-<p >1、L1、L2都能扩增得到1300bp的产物，鉴定的重组载体含有外源基因mlrA </p>
-<p >2、M是100bp&nbsp;Ladder&nbsp;Marker </p>
-<p >将经过PCR鉴定的含有重组载体pSB1C3-A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5），合成的mlrA基因序列与设计序列一致。 </p>
-<p ><img width="584" height="334" src="5_wps5268.tmp.png" ><img width="584" height="240" src="5_wps5278.tmp.png" >&nbsp;</p>
-<p >图5&nbsp;测序结果与设计序列比对 </p>
-<p >&nbsp;</p>
-<h3 ><img width="155" height="58" src="6_wps9D1F.tmp.png" >6、引物合成 </h3>
-<table >
-  <tr >
-    <td width="1038" valign="center" rowspan="3" ><p >&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; </p>
-      <table >
-        <tr >
-          <td width="1023" valign="center" rowspan="2" ><p >吉林省库美生物科技有限公司引物合成订购单 </p></td>
-        </tr>
-        <tr ></tr>
-      </table>
-      <p >&nbsp;</p></td>
-  </tr>
-  <tr ></tr>
-  <tr ></tr>
-</table>
-<p >&nbsp;</p>
-<table >
-  <tr >
-    <td width="50" valign="center" ><p >引物编号 </p></td>
-    <td width="456" valign="center" ><p >序列，(5'&nbsp;to&nbsp;3') </p></td>
-    <td width="57" valign="center" ><p >碱基数（bp） </p></td>
-    <td width="57" valign="center" ><p >合成总量(OD) </p></td>
-    <td width="47" valign="center" ><p >分装管数 </p></td>
-    <td width="56" valign="center" ><p >纯化方式 </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A101 </p></td>
-    <td width="456" valign="center" ><p >CCGGAATTCATGCGAGAATTTGTTCGACAACGACCATTATTATGTTTTTATGTTTTAGCT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A102 </p></td>
-    <td width="456" valign="center" ><p >CTCGTAAAGCATGAGCAGCTAAAGCAATTAAAATAGCTAAAACATAAAAACATAATA </p></td>
-    <td width="57" valign="center" ><p >57 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A103 </p></td>
-    <td width="456" valign="center" ><p >AGCTGCTCATGCTTTACGAGCTATGTCACCAACTCCATTAGATCCAATGTTTAAAATGTT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A104 </p></td>
-    <td width="456" valign="center" ><p >AGCAGTAATAATATTTAAATGAGCATGAGTTTCTTGTAACATTTTAAACATTGGATCTAA </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A105 </p></td>
-    <td width="456" valign="center" ><p >CTCATTTAAATATTATTACTGCTGTTCGATCAACTTTTGAATATCCAGGAGCTTATACTT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A106 </p></td>
-    <td width="456" valign="center" ><p >CAGCAAACATTGGAGCAGCTGGAAATAATAATAAAGTATAAGCTCCTGGATATTCA </p></td>
-    <td width="57" valign="center" ><p >56 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A107 </p></td>
-    <td width="456" valign="center" ><p >GCTGCTCCAATGTTTGCTGCTTTAATTGCTACTGGAATTGGATATGGACAAGCTGGATTT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A108 </p></td>
-    <td width="456" valign="center" ><p >TCGCCATGGAGCACATCGTGATAATAATTCTCGAAATCCAGCTTGTCCATATCCAA </p></td>
-    <td width="57" valign="center" ><p >56 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A109 </p></td>
-    <td width="456" valign="center" ><p >ACGATGTGCTCCATGGCGATCACCAGTTTCATGGCGACAAGGAGTTACTGTTATTGCTGT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A110 </p></td>
-    <td width="456" valign="center" ><p >CATAATTCCAGTTAAAGCAAAAAAAGCTAAAAAACAAACAGCAATAACAGTAACTCCTTG </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A111 </p></td>
-    <td width="456" valign="center" ><p >TTTTGCTTTAACTGGAATTATGTGGGTTCAAACTTATTTATATGCTCCACCAGGAACTTT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A112 </p></td>
-    <td width="456" valign="center" ><p >CAACTGGATCTGATCCATATCGTAAAAAAGTTCGATCTAAAGTTCCTGGTGGAGCATATA </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A113 </p></td>
-    <td width="456" valign="center" ><p >CGATATGGATCAGATCCAGTTGCTATTTATGTTATGTTAGCTGCTTCATTATTATTATCA </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A114 </p></td>
-    <td width="456" valign="center" ><p >ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAGCAGCT </p></td>
-    <td width="57" valign="center" ><p >44 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A201 </p></td>
-    <td width="456" valign="center" ><p >ATTAGAAGAATTAGGATGGCGAGGATTTGCTTTACCACAATTATTAAAAAAATTTGATCC </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A202 </p></td>
-    <td width="456" valign="center" ><p >CACCACATAATTCCTAAAATAACAGCAGCAGTTAATGGATCAAATTTTTTTAATAATTGT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A203 </p></td>
-    <td width="456" valign="center" ><p >TGTTATTTTAGGAATTATGTGGTGGGCTTGGCATTTACCACGAGATTTACCAACTTTATT </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A204 </p></td>
-    <td width="456" valign="center" ><p >AACTGACCAAGCAGCTCCTGGAGCTCCTGAAAATAAAGTTGGTAAATCTCGTGGT </p></td>
-    <td width="57" valign="center" ><p >55 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A205 </p></td>
-    <td width="456" valign="center" ><p >AGGAGCTGCTTGGTCAGTTATTGTTAAACAATTAGTTATTACTCCAGGATTTATTGCTTC </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A206 </p></td>
-    <td width="456" valign="center" ><p >CCTAATTTATTACAAACAAAAACAGCAATAATAGTTGAAGCAATAAATCCTGGAGTAATA </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A207 </p></td>
-    <td width="456" valign="center" ><p >TGTTTTTGTTTGTAATAAATTAGGAGGATCAATGTGGGGAGGAGTTTTAACTCATGC </p></td>
-    <td width="57" valign="center" ><p >57 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A208 </p></td>
-    <td width="456" valign="center" ><p >CATTCAGCAGTAACATTAACTCCTAATTCATTATGAATAGCATGAGTTAAAACTCCTCCC </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A209 </p></td>
-    <td width="456" valign="center" ><p >AGGAGTTAATGTTACTGCTGAATGGGCTCCAACTGTTGCTGGATTAGGATGGCGA </p></td>
-    <td width="57" valign="center" ><p >55 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A210 </p></td>
-    <td width="456" valign="center" ><p >ACTAATCCAATAGCAACAGCAAATTCAATTAAATCCCATGGTCGCCATCCTAATCCAGCA </p></td>
-    <td width="57" valign="center" ><p >60 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A211 </p></td>
-    <td width="456" valign="center" ><p >ATTTGCTGTTGCTATTGGATTAGTTTTAATTTGTGGACGATCATTAGGAGCTGCTTC </p></td>
-    <td width="57" valign="center" ><p >57 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A212 </p></td>
-    <td width="456" valign="center" ><p >GTGGAACATTTCCCCAAGCTAATCGAGCATTATCTGGTGAAGCAGCTCCTAATGATCGT </p></td>
-    <td width="57" valign="center" ><p >59 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A213 </p></td>
-    <td width="456" valign="center" ><p >TTAGCTTGGGGAAATGTTCCACCAAAATTACCAGGAGGAGTTGGAGATAAATCAGGAGC </p></td>
-    <td width="57" valign="center" ><p >59 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A214 </p></td>
-    <td width="456" valign="center" ><p >AAGACGTCCTATTAAGCATTAGCTCCTGATTTATCTCCAACTC </p></td>
-    <td width="57" valign="center" ><p >43 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A1 </p></td>
-    <td width="456" valign="center" ><p >CCGGAATTCATGCGAGAATTTGTTCG </p></td>
-    <td width="57" valign="center" ><p >26 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A2 </p></td>
-    <td width="456" valign="center" ><p >ATTCTTCTAATAATGGTCCTGGTGATAATAATAATGAAG </p></td>
-    <td width="57" valign="center" ><p >39 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A3 </p></td>
-    <td width="456" valign="center" ><p >ATTAGAAGAATTAGGATGGCGAGGATTTGC </p></td>
-    <td width="57" valign="center" ><p >30 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A4 </p></td>
-    <td width="456" valign="center" ><p >AAGACGTCCTATTAAGCATTAGCTCCTG </p></td>
-    <td width="57" valign="center" ><p >28 </p></td>
-    <td width="57" valign="center" ><p >1 </p></td>
-    <td width="47" valign="center" ><p >1 </p></td>
-    <td width="56" valign="center" ><p >PAGE </p></td>
-  </tr>
-  <tr >
-    <td width="50" valign="center" ><p >A5 </p></td>
-    <td width="456" valign="center" ><p >AACTGCAGCTATTAAGCATTAGCTCCTG </p></td>
-    <td width="57" valign="center" ><p >28 </p></td>
-    <td width="57" valign="center" ><p >&nbsp;</p></td>
-    <td width="47" valign="center" ><p >&nbsp;</p></td>
-    <td width="56" valign="center" ><p > </p></td>
-  </tr>
-</table>
-</div>
 </body>
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