Team:Jilin China/RESULT

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<h3 >2、Mixing of primers</h3>
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<h3 id="coop">2、Mixing of primers</h3>
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<p >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>
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<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>
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<p >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>
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<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>
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<p >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>
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<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>
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     <td width="67"><p align="center"><strong>Group</strong></p></td>
     <td width="67"><p align="center"><strong>Group</strong></p></td>
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<h3 >3、DA-PCR </h3>
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<h3 id="coop">3、DA-PCR </h3>
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<p >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>
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<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>
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<p>Procedure of DA-PCR： <br>
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<p id="coop">Procedure of DA-PCR： <br>
   Mixed primer solutions                   5μl<br>
   Mixed primer solutions                   5μl<br>
   Pfu DNA Polymerase  2.5U              0.5μl     <br>
   Pfu DNA Polymerase  2.5U              0.5μl     <br>
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   72℃         1min<br>
   72℃         1min<br>
   72℃        10min<br>
   72℃        10min<br>
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   4℃preservation<br>
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   4℃        preservation<br>
   End<br>
   End<br>
   DA-PCR splicing reaction product was  detected in 2% agarose gel electrophoresis,<br>
   DA-PCR splicing reaction product was  detected in 2% agarose gel electrophoresis,<br>
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75V electrophoress for 1h。 </p>
75V electrophoress for 1h。 </p>
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<p ><img src="https://static.igem.org/mediawiki/2014/8/84/Simonsong-result-1.png" ></p>
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<div style="text-align:center"><p id="coop"><img src="https://static.igem.org/mediawiki/2014/8/84/Simonsong-result-1.png" ></p></div>
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<p>Figure1 DA-PCR  splicing results <br>
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<p id="coop" align="center">Figure1 DA-PCR  splicing results <br></P>
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  Illustration of  the outcome:<br>
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<p id="coop"> Illustration of  the outcome:<br>
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   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.</p>
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   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.<br>
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2、A5 could  obtainintermediates(Block5) which were made by four single-stranded  oligonucleotides splicedtogether.
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2、A5 could  obtainintermediates(Block5) which were made by four single-stranded  oligonucleotides splicedtogether.</p>
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<h3 >4、OE-PCR </h3>
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<h3 id="coop">4、OE-PCR </h3>
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<p >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>
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<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>
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<p>Block1                       1μl<br>
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<p id="coop">Block1                       1μl<br>
   Block2                       1μl<br>
   Block2                       1μl<br>
   Block3                       1μl<br>
   Block3                       1μl<br>
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  Sterilized  ultrapure water   to 50ul<br>
  Sterilized  ultrapure water   to 50ul<br>
   Procedure of OE-PCR： <br>
   Procedure of OE-PCR： <br>
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   94℃         2min<br>        94℃         30S<br>
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   94℃         2min<br>         
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94℃         30S<br>
   47℃         30S    20 cycles<br>
   47℃         30S    20 cycles<br>
   72℃        2min<br>
   72℃        2min<br>
   72℃       10min<br>
   72℃       10min<br>
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     4℃preservation<br>
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 4℃        preservation<br>
   End<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>
   The full-length gene  spliced by OE-PCR was detected by a 1% agarose gel electrophoresis, specific  programs are as follows<br>
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  100bp  Marker              5μl   spotted directly<br>
  100bp  Marker              5μl   spotted directly<br>
   80V electrophoresis for  1h.</p>
   80V electrophoresis for  1h.</p>
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<p ><img src="https://static.igem.org/mediawiki/2014/2/24/Simonsong-result-2.png" ></p>
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<<p>Illustration of  the outcome:</p>
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<p id="coop">Illustration of  the outcome:</p>
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<ol>
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<ol id="coop">
   <li>L1,  L2, L3 could get mlrA genome which were made by five single-stranded  oligonucleotides splicedtogether.</li>
   <li>L1,  L2, L3 could get mlrA genome which were made by five single-stranded  oligonucleotides splicedtogether.</li>
   <li>Wide  strip of the tape at 100bp was caused by excessive amount of amplification  primers A1 and A4.</li>
   <li>Wide  strip of the tape at 100bp was caused by excessive amount of amplification  primers A1 and A4.</li>
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<h3 id="coop">5、Constructing and sequencing of subcoloning vector</h3>
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<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>
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<h3 >5、Constructing and sequencing of subcoloning vector</h3>
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<p>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>
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   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>
   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>
   Gene or plasmid                       10μl<br>
   Gene or plasmid                       10μl<br>
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<p ><img  src="https://static.igem.org/mediawiki/2014/0/0d/Simonsong-result-3.png" >&nbsp;</p>
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<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>
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<p >Fig3.Reconstructed vector pSB1C3-A </p>
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<p id="coop" align="center">Fig3.Reconstructed vector pSB1C3-A </p>
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<p >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>
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<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>
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<p ><img  src="https://static.igem.org/mediawiki/2014/6/63/Simonsong-result-4.png" ></p>
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<div style="text-align:center"><p id="coop"><img  src="https://static.igem.org/mediawiki/2014/6/63/Simonsong-result-4.png" ></p></div>
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<p >&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>
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<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>
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<p >Results: </p>
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<p id="coop">Results: </p>
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<p >1.L1 and L2 is 1300bp and it confirms that the reconstructed vector has mlrA on it. </p>
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<p id="coop">1.L1 and L2 is 1300bp and it confirms that the reconstructed vector has mlrA on it. </p>
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<p >2.M is the 100bp Ladde Marker. </p>
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<p id="coop">2.M is the 100bp Ladde Marker. </p>
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<p >Sequencing result shows that the sequence of synthetic mlrA gene is exactly same as sequence of designed sequence. </p>
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<p id="coop">Sequencing result shows that the sequence of synthetic mlrA gene is exactly same as sequence of designed sequence. </p>
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<p ><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>
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<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>
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<p > Fig5.Comparison of result of sequencing and designed sequence </p>
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<p id="coop"align="center" > Fig5.Comparison of result of sequencing and designed sequence </p>
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<p id="coop">&nbsp;</p>
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Latest revision as of 03:59, 18 October 2014

Welcome!
Team Jilin China

Retrieved from "http://2014.igem.org/Team:Jilin_China/RESULT"
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:

  1. L1, L2, L3 could get mlrA genome which were made by five single-stranded oligonucleotides splicedtogether.
  2. 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