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From 2014.igem.org

Contents 1 Protocol 1.1 Transformation 1.2 Plasmid extraction 1.3 Restriction enzyme digestion 1.4 Ligation 1.5 Microplate reader 1.6 Flow cytometer 1.7 Biobrick structure: 1.8 Note：

Protocol

Transformation

&nbsp&nbsp&nbsp&nbsp6μL plasmid solution (about 50ng) is well mixed with 25μL competent DH5α solution and then incubated for 30min on ice.

&nbsp&nbsp&nbsp&nbspAdd 500μL LB medium and incubate in 37℃ for 45min-1h.

&nbsp&nbsp&nbsp&nbspCentrifuge at 10000g for 30s, remove 400μL LB, and resuspend by voltex. Spread the culture on a LB plate with corresponding resistance. A 12h cultivation at 37℃ may be fairly enough to obtain the strain you need.

&nbsp&nbsp&nbsp&nbsp（If the plasmid for transformation is prepared from ligation, following steps are necessary.）

&nbsp&nbsp&nbsp&nbspAfter 12h cultivation, inoculate 20 grown single colonies into another LB plate with same resistance, and number them. (PATCH)

&nbsp&nbsp&nbsp&nbspAfter more than 7h of incubation a colony PCR is conducted, using the colony respectively as template and standard VF and VR oligo as primers in the system, in which the first step should be 94℃ for 10min to release the inner plasmid.

&nbsp&nbsp&nbsp&nbspPick out positive strains whose PCR product contains the target gene.

Plasmid extraction

&nbsp&nbsp&nbsp&nbspInoculate the strain with plasmid into 5mL LB with proper resistance, incubating for 12-18h.

&nbsp&nbsp&nbsp&nbspCollect the plasmid by Hipure plasmid Micro Kit (Bought from Magen), following the product instruction.

Restriction enzyme digestion

&nbsp&nbsp&nbsp&nbspSolution containing 1ng DNA was prepared for digestion, which follows the method of instruction of Thermo FastDigest restriction enzyme, following the product instruction.

&nbsp&nbsp&nbsp&nbspTarget DNA was isolated from digested DNA solution by electrophoresis in 1.2% agarose, 105V for 37min.

&nbsp&nbsp&nbsp&nbspA gel extraction is conducted with HiPure Gel Pure DNA Micro Kit (bought from Magen) to purify digested sequences, following the product instruction.

Ligation

The procedure of Ligation of target sequences is following the instruction of Takara T4 ligase, following the product instruction.

Microplate reader

&nbsp&nbsp&nbsp&nbspBacteria strains containing biobricks were inoculated into a 12 mL tube with 1.5mL LB of corresponding resistance and incubated at 37°C, 200 rpm shaking for 18 hours.

&nbsp&nbsp&nbsp&nbspThe grown cultures were then diluted 1:100 into another 12 mL tube with 1.5 ml of LB medium and incubated in the same condition as before for 3 hours.

&nbsp&nbsp&nbsp&nbspEach strain was sampled 200μL for 3 parallel wells into a flat-bottom 96-well microplate (sampling time approximately 15min).

&nbsp&nbsp&nbsp&nbspGFP fluorescence and O.D.600 were measured by Synergy H1 Hybrid Multi-Mode Microplate Reader. 15s linear shake (amplitude 3mm) and 10s waiting are conducted before to mix the culture.

&nbsp&nbsp&nbsp&nbspEach strain is sampled was essayed thrice and each sample is parallelly measured in 3 wells.

Flow cytometer

&nbsp&nbsp&nbsp&nbspBacteria strains containing biobricks were inoculated into a 12 mL tube with 1.5mL LB of corresponding resistance and incubated at 37°C, 200 rpm shaking for 18 hours.

&nbsp&nbsp&nbsp&nbspThe grown cultures were then diluted 1:100 into another 15 mL Falcon tube with 5 ml LB medium and incubated in the same condition as before for 3-4 hours (O.D.600 = 0.8-1.0).

&nbsp&nbsp&nbsp&nbsp150 μL of the grown cultures were than centrifuged at 8000 rpm for 2 minutes.

&nbsp&nbsp&nbsp&nbspAfter centrifugation the supernatant was discarded and the sediments were washed by PBS (pH=7.4) for two times.

&nbsp&nbsp&nbsp&nbspAdded 600 μL of PBS (pH=7.4) into the sediments and fully resuspend.

&nbsp&nbsp&nbsp&nbspThe solution were transferred to the flow cytometer sample tube, ready for essay.

&nbsp&nbsp&nbsp&nbspTest GFP fluorescence of each cell, using wt DH5α as control to optimize parameters.

Biobrick structure:

&nbsp&nbsp&nbsp&nbspThe required plasmid was transformed in E coli. DH5α

&nbsp&nbsp&nbsp&nbspThe measured devices are

&nbsp&nbsp&nbsp&nbspGFP:

&nbsp&nbsp&nbsp&nbsp&nbsp&nbspBBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector

&nbsp&nbsp&nbsp&nbsp&nbsp&nbspBBa_J23101 + BBa_E0240 (B0032-E0040-B0015) in the pSB1C3 vector

&nbsp&nbsp&nbsp&nbsp&nbsp&nbspBBa_J23115 + BBa_E0240 (B0032-E0040-B0015) in the pSB1C3 vector

&nbsp&nbsp&nbsp&nbspRFP: <p>&nbsp&nbsp&nbsp&nbsp&nbsp&nbspBBa_I23101 + BBa_K516032 (B0032-E1010-B0015) in the pSB3K3 vector

&nbsp&nbsp&nbsp&nbsp&nbsp&nbspBBa_J23101 + BBa_K516032 (B0032-E1010-B0015) in the pSB1C3 vector

Note：

1. The devices BBa_I20260 (J23101-B0032-E0040-B0015) in the pSB3K3 vector and BBa_I23101 + BBa_K516032 (B0032-E1010-B0015) in the pSB3K3 vector are used as reference in this experiment.

2. BBa_J23115 distributed in this year’s distribution kit was used in the experiment, but since the sequencing result of J23115 with RFP shows that some base pairs did not match with the standard BBa_J23115 (Even repeated for 3 times), we stopped the measurement of J23115 with RFP.