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.

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Team:Shanghaitech/protocal