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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