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Team:Shanghaitech/protocal

Team:Shanghaitech - 2017.igem.org

Team:Shanghaitech

Shanghaitech

Introduction

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I. PCR amplification of fragment

Pipet the 50 uL reaction system into a PCR tube
25 uL Tiangen 2×Taq PCR MasterMix(with loading dye)
1 uL plasmid template
2.5 uL forward primer
2.5 uL reverse primer
19 uL ddH2O
PCR cycle
94℃ 3 min
30 cycles
94℃ 30 sec
55℃ 30 sec
72℃ 1 min
72℃ 5 min
Products are purified using 1% agarose gel and TIANgel Mini DNA Purification Kit.

II. DNA digestion of two parts

1. Pipet the following into a PCR tube
20uL fragment digestion system
16 uL DNA(PCR product)
2 uL 10×H buffer
1 uL Takara EcoRI
1 uL Takara
20 uL vector part digestion system
16 uL DNA(part plasmid)
2 uL 10×M buffer
1 uL Takara EcoRI
1 uL Takara XbaI
2. Incubation
Incubate the tubes at 16℃ overnight (≥12h)
3. Purification
Fragment is purified using TIANquick Midi Purification Kit, and the vector is purified using 1% agarose gel and TIANgel Mini DNA Purification Kit.
Concentrations of digestion products are tested after purification.

III. Ligation of two parts

1. Pipet the following system into a PCR tube
10 uL reaction system(for higher concentrations)
8 uL total DNA(vector and fragment with molar ratio around 1:5)
1 uL 10×ligation buffer
1 uL Takara T4 DNA Ligase
20 to 50 uL reaction system(for lower concentrations, both <10ng/uL)
Let total volume be y uL
(0.9y-1) uL total DNA (molar ratio = 1:5)
0.1y uL 10×ligation buffer
1uL Takara T4 DNA Ligase
2. Incubation
Incubate the tubes at 16℃ overnight (≥12h)
3. Transformation
Add 1uL of the mixture directly without purification into competent DH5α cell mixture.

IV.Fluorescence Expression Measurements

Cells were prepared for plate reader experiments as follows: strains were grown overnight and then reseeded in a 1:1000 dilution into fresh media containing ampicillin and spectinomycin resistance. The dilution was allowed to grow for about 4 h until it reached OD ≈ 0.1. Then, the cell culture were distributed into a 96-well plate (180 μl final volume per well) and induced with a range of AHL concentrations (1:10 serial dilutions were done from 1 × 10–4 M to 1 × 10–10 M or 1 × 10–14 M). AHLs stocks were dissolved in DMSO, resulting in a max DMSO concentration of 1% in LB. After induction, the cells were allowed to grow for 3 h until OD ≈ 0.5 at which point they were measured with a Tecan infinite M200Pro for OD600 as well as GFP fluorescence with the following fixed settings: no top, fixed gain of 61, excitation of 485 nm, emission of 520 nm, and Z-position of 19500. All GFP measurements were normalized by dividing their raw values by the OD of that well to give a “per-cell” measurement and account for slight differences in growth rates. For Figure 6, the same methods were used, except gain regulation was used to find an optimal gain for each individual experiment, and RFP levels were measured with an excitation of 580 nm and an emission of 620 nm.

V.E.Coli荧光读取酶标仪Protocol Version 0.1

此版本仅包括应知应会的程序设置流程,具体使⽤用⽅方式如加样时的对照组选择等将于后续奉上
使⽤用前请注意,每次加样体积应保持⼀一致(推荐每孔100-200μl,不不要加太满)
为尽可能减⼩小误差,每个样最好加三个孔
程序设置:
注意:操作酶标仪及电脑均禁⽌止使⽤用⼿手套!
1. 开机,选择酶标仪的控制软件。
2. 点击“Plate”,选择setting
3. 依次选择:
1. Monochromator,分光计模式。
2. FL,荧光模式
3. Endpoint/Kinetic(取决于你的需要)
1.Endpoing模式:
选择好波⻓长
板⼦子的类型,默认即是我们所⽤用的oppque,根据你是否加盖,在lidded项勾选
读取区域,框选你所需读取的井 PMT & Optics⽆无需调整
Shake最好⽤用linear模式,读取前⽬目前推荐摇5s,之后会视情况统⼀一 读取暂定从上⾯面,不不勾选从下⾯面读取的框。
2.Knietics模式:
选择好波⻓长
板⼦子的类型,默认即是我们所⽤用的oppque,根据你是否加盖,在lidded项勾选
读取区域,框选你所需读取的井 选择读数总时间与两次读数间隔,会⾃自动计算读取总数。这个间隔还需之后确定
PMT & Optics⽆无需调整
Shake最好⽤用linear模式,读取前⽬目前推荐开始和两次读取间摇5s,之后会视情况统⼀一 读取暂定从上⾯面,不不勾选从下⾯面读取的框。

Appendix

pcons+lacI+ter

TTGACGGCTA GCTCAGTCCT AGGTACAGTG CTAGCAAAGA GGAGAAATAC TAGATGGTGA ATGTGAAACC AGTAACGTTA TACGATGTCG CAGAGTATGC CGGTGTCTCT TATCAGACCG TTTCCCGCGT GGTGAACCAG GCCAGCCACG TTTCTGCGAA AACGCGGGAA AAAGTGGAAG CGGCGATGGC GGAGCTGAAT TACATTCCCA ACCGCGTGGC ACAACAACTG GCGGGCAAAC AGTCGTTGCT GATTGGCGTT GCCACCTCCA GTCTGGCCCT GCACGCGCCG TCGCAAATTG TCGCGGCGAT TAAATCTCGC GCCGATCAAC TGGGTGCCAG CGTGGTGGTG TCGATGGTAG AACGAAGCGG CGTCGAAGCC TGTAAAGCGG CGGTGCACAA TCTTCTCGCG CAACGCGTCA GTGGGCTGAT CATTAACTAT CCGCTGGATG ACCAGGATGC CATTGCTGTG GAAGCTGCCT GCACTAATGT TCCGGCGTTA TTTCTTGATG TCTCTGACCA GACACCCATC AACAGTATTA TTTTCTCCCA TGAAGACGGT ACGCGACTGG GCGTGGAGCA TCTGGTCGCA TTGGGTCACC AGCAAATCGC GCTGTTAGCG GGCCCATTAA GTTCTGTCTC GGCGCGTCTG CGTCTGGCTG GCTGGCATAA ATATCTCACT CGCAATCAAA TTCAGCCGAT AGCGGAACGG GAAGGCGACT GGAGTGCCAT GTCCGGTTTT CAACAAACCA TGCAAATGCT GAATGAGGGC ATCGTTCCCA CTGCGATGCT GGTTGCCAAC GATCAGATGG CGCTGGGCGC AATGCGCGCC ATTACCGAGT CCGGGCTGCG CGTTGGTGCG GATATCTCGG TAGTGGGATA CGACGATACC GAAGACAGCT CATGTTATAT CCCGCCGTTA ACCACCATCA AACAGGATTT TCGCCTGCTG GGGCAAACCA GCGTGGACCG CTTGCTGCAA CTCTCTCAGG GCCAGGCGGT GAAGGGCAAT CAGCTGTTGC CCGTCTCACT GGTGAAAAGA AAAACCACCC TGGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC CGATTCATTA ATGCAGCTGG CACGACAGGT TTCCCGACTG GAAAGCGGGC AGTAATAATA CTAGAGCCAG GCATCAAATA AAACGAAAGG CTCAGTCGAA AGACTGGGCC TTTCGTTTTA TCTGTTGTTT GTCGGTGAAC GCTCTCTACT AGAGTCACAC TGGCTCACCT TCGGGTGGGC CTTTCTGCGT TTATA

plac+lasI+ter+pcons+lacI+ter

CAATACGCAA ACCGCCTCTC CCCGCGCGTT GGCCGATTCA TTAATGCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC GCAACGCAAT TAATGTGAGT TAGCTCACTC ATTAGGCACC CCAGGCTTTA CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA ATTTCACACA TACTAGAGAA AGAGGAGAAA TACTAGATGA TCGTTCAGAT CGGTCGTCGT GAAGAGTTCG ACAAAAAACT GCTGGGTGAA ATGCACAAAC TGCGTGCTCA GGTTTTCAAA GAACGTAAAG GTTGGGACGT TTCCGTTATC GACGAAATGG AAATCGACGG TTACGACGCT CTGTCCCCGT ACTACATGCT GATCCAGGAA GACACCCCGG AAGCTCAGGT TTTCGGTTGC TGGCGTATCT TCGACACCAC CGGTCCGTAC ATGCTGAAAA ACACCTTCCC GGAACTGCTG CACGGTAAAG AAGCTCCGTG CTCCCCGCAC ATCTGGGAAC TGTCCCGTTT CGCTATCAAC TCCGGTCAGA AAGGTTCCCT GGGTTTCTCC GACTGCACCC TGGAAGCTAT GCGTGCTCTG GCTCGTTACT CCTTGCAGAA CGACATCCAG ACCCTGGTTA CCGTTACCAC CGTTGGTGTT GAAAAAATGA TGATCCGTGC TGGTCTGGAC GTTTCCCGTT TCGGTCCGCA CCTGAAAATC GGTATCGAAC GTGCTGTTGC TCTGCGTATC GAACTGAACG CTAAAACCCA GATCGCTCTG TACGGTGGTG TTCTGGTTGA ACAGCGTCTG GCTGTTTCCC ATCATCATCA TCATCATTAA TAATACTAGA GCCAGGCATC AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT TGTTTGTCGG TGAACGCTCT CTACTAGAGT CACACTGGCT CACCTTCGGG TGGGCCTTTC TGCGTTTATA TACTAGAGTT GACGGCTAGC TCAGTCCTAG GTACAGTGCT AGCAAAGAGG AGAAATACTA GATGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCAC AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGC ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCG TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATC TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGA CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATC TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGG CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAG CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAA TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTC GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC CAGGCGGTGA AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TAATAATACT AGAGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGTGAACGC TCTCTACTAG AGTCACACTG GCTCACCTTC GGGTGGGCCT TTCTGCGTTT ATA

plac+rhlL+ter+pcons+lacI+ter

CAATACGCAA ACCGCCTCTC CCCGCGCGTT GGCCGATTCA TTAATGCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC GCAACGCAAT TAATGTGAGT TAGCTCACTC ATTAGGCACC CCAGGCTTTA CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA ATTTCACACA TACTAGAGAA AGAGGAGAAA TACTAGATGA TCGAACTGCT GTCCGAATCC CTGGAAGGTC TGTCCGCTGC TATGATCGCT GAACTGGGTC GTTACCGTCA CCAGGTTTTC ATCGAAAAAC TGGGTTGGGA CGTTGTTTCC ACCTCCCGTG TTCGTGACCA GGAGTTCGAC CAGTTCGACC ACCCGCAGAC CCGTTACATC GTTGCTATGT CCCGTCAGGG TATCTGCGGT TGCGCTCGTC TGCTGCCGAC CACCGACGCT TACCTGCTGA AAGACGTTTT CGCTTACCTG TGCTCCGAAA CCCCGCCGTC CGACCCGTCC GTTTGGGAAC TGTCCCGTTA CGCTGCTTCC GCTGCTGACG ACCCGCAGCT GGCTATGAAA ATCTTCTGGT CCTCCCTCCA GTGCGCTTGG TACCTGGGTG CTTCCTCCGT TGTTGCTGTT ACCACCACCG CTATGGAACG TTACTTCGTT CGTAACGGTG TTATCCTCCA GCGTCTGGGT CCGCCGCAGA AAGTTAAAGG TGAAACCCTG GTTGCTATCT CCTTCCCGGC TTACCAGGAA CGTGGTCTGG AAATGCTGCT GCGTTACCAC CCGGAATGGC TCCAGGGTGT TCCGCTGTCC ATGGCTGTTC ATCATCATCA TCATCATTAA TAATACTAGA GCCAGGCATC AAATAAAACG AAAGGCTCAG TCGAAAGACT GGGCCTTTCG TTTTATCTGT TGTTTGTCGG TGAACGCTCT CTACTAGAGT CACACTGGCT CACCTTCGGG TGGGCCTTTC TGCGTTTATA TACTAGAGTT GACGGCTAGC TCAGTCCTAG GTACAGTGCT AGCAAAGAGG AGAAATACTA GATGGTGAAT GTGAAACCAG TAACGTTATA CGATGTCGCA GAGTATGCCG GTGTCTCTTA TCAGACCGTT TCCCGCGTGG TGAACCAGGC CAGCCACGTT TCTGCGAAAA CGCGGGAAAA AGTGGAAGCG GCGATGGCGG AGCTGAATTA CATTCCCAAC CGCGTGGCAC AACAACTGGC GGGCAAACAG TCGTTGCTGA TTGGCGTTGC CACCTCCAGT CTGGCCCTGC ACGCGCCGTC GCAAATTGTC GCGGCGATTA AATCTCGCGC CGATCAACTG GGTGCCAGCG TGGTGGTGTC GATGGTAGAA CGAAGCGGCG TCGAAGCCTG TAAAGCGGCG GTGCACAATC TTCTCGCGCA ACGCGTCAGT GGGCTGATCA TTAACTATCC GCTGGATGAC CAGGATGCCA TTGCTGTGGA AGCTGCCTGC ACTAATGTTC CGGCGTTATT TCTTGATGTC TCTGACCAGA CACCCATCAA CAGTATTATT TTCTCCCATG AAGACGGTAC GCGACTGGGC GTGGAGCATC TGGTCGCATT GGGTCACCAG CAAATCGCGC TGTTAGCGGG CCCATTAAGT TCTGTCTCGG CGCGTCTGCG TCTGGCTGGC TGGCATAAAT ATCTCACTCG CAATCAAATT CAGCCGATAG CGGAACGGGA AGGCGACTGG AGTGCCATGT CCGGTTTTCA ACAAACCATG CAAATGCTGA ATGAGGGCAT CGTTCCCACT GCGATGCTGG TTGCCAACGA TCAGATGGCG CTGGGCGCAA TGCGCGCCAT TACCGAGTCC GGGCTGCGCG TTGGTGCGGA TATCTCGGTA GTGGGATACG ACGATACCGA AGACAGCTCA TGTTATATCC CGCCGTTAAC CACCATCAAA CAGGATTTTC GCCTGCTGGG GCAAACCAGC GTGGACCGCT TGCTGCAACT CTCTCAGGGC CAGGCGGTGA AGGGCAATCA GCTGTTGCCC GTCTCACTGG TGAAAAGAAA AACCACCCTG GCGCCCAATA CGCAAACCGC CTCTCCCCGC GCGTTGGCCG ATTCATTAAT GCAGCTGGCA CGACAGGTTT CCCGACTGGA AAGCGGGCAG TAATAATACT AGAGCCAGGC ATCAAATAAA ACGAAAGGCT CAGTCGAAAG ACTGGGCCTT TCGTTTTATC TGTTGTTTGT CGGTGAACGC TCTCTACTAG AGTCACACTG GCTCACCTTC GGGTGGGCCT TTCTGCGTTT ATA

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