Difference between revisions of "Team:SKLMT-China/Notebook Overview"

Team registration and brainstorm

During this time, we registrated the team SKLMT-China in iGEM. In order to verify the feasibility of our project, we discussed with professors and experts and did some human practice work.

Find promoters with various strength in the light of transcriptom references. Take the sequence from the upstream of a specific gene.

design the PAGE-purified oligonucleotides used to amplify the chloramphenicol

Amplify the chloramphnicol and promoter segment and add vector homology arms via series PCR. Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH₂O. Quantify the elution using a NanoDrop UV spectrophotometer, store them at 4℃。

In order to obtain an promoter library suitable for cloning, promoter sequence flanked by homology arms was added a chloramphenicol selection marker. This sequence was synthesized by Sangon.

TABLE 2 Oligonucleotides used for PCR of the DNA segment containing the promoter and chloramphenicol selective marker

Set up the PCR reaction using pR6K-cm-ccdB as a template. Typically, set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA.

Carry out the PCR in a thermal cycle following the instructions below:

·Add vector homology arms to the PCR products purified.

·Use the purified PCR products from last step as a template. Use the PAGE-purified oligonucleotides ,Syn-promoter-3 and promoter-4 (Table3), to amplify the cm-promoter.

TABLE 3 Oligonucleotides used for PCR of the DNA segment containing the promoter and chloramphenicol selective marker

·Use the purified PCR products from step2 as a template. set up the PCR reaction (50ul in total) to obtain sufficient amounts of DNA

Carry out the PCR in a thermal cycle following the instructions below:

·Purify the PCR products using the TianGen PCR Purification Kit and elute into 30ul of autoclaved ddH₂O. Quantify the elution using a NanoDrop UV spectrophotometer.

Transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into GB05 red gyrA462 via electroporation. Do Mini-Prep and screen correct plasmid by restriction analysis

construct the plasmid pBBR1-kan-cm-promoter-firefly via liner circular recombination in GB05 red gyrA462.

Do Mini-Prep and screen correct recombinants by restriction analysis.

Transform the plasmid pBBR1-kan-amp-ccdb-BAD-GFP-firefly into P.fluorescence pf-5 via electroporation.

·Use a syringe needle (25-Gauge 5/8, 0.5 mm × 16 mm) to puncture the cap of a 1.5-ml microcentrifuge tube for aeration and add 1.0 ml LB. Inoculate the medium in the 1.5-ml tube with the several colonies of P. fluorescence pf-5 cells and incubate at 30 °C overnight with shaking at 950 r.p.m. in an Eppendorf thermomixer.

·Puncture the cap of a 1.5-ml microcentrifuge tube, add 1.4 ml of LB inoculating with 40 ul of the overnight culture.

·Incubate the mixture at 30 °C for 2.5h with shaking at 950 r.p.m. in an Eppendorf thermomixer. At this stage, the OD600 should be between 0.35 and 0.40.

· Put the cells on ice (and keep them on the ice whenever possible).

·Spin the cells down at 9,000 rpm for 30 sec in the cooling centrifuge at 2°C.

·Discard the supernatant by decanting. Discard as much supernatant as possible.

· Resuspend the pellet in 1 ml of ice-cold 10% sucrose buffer (containing 2mM HEPES) by pipetting.

· Spin the cells at 10,000 rpm for 30 sec in the cooling centrifuge at 2°C.

·Discard the supernatant by decanting. Discard as much supernatant as possible.

· Resuspend the pellet in 1 ml of ice-cold sucrose buffer (containing 2mM HEPES) by pipetting.

· Spin the cells down at 11,000 rpm for 30 sec in the cooling centrifuge at 2°C.

· Discard the supernatant by decanting and leave about 30 µl.

· Add DNA to the cells and pipette the mixture into the chilled 1 mm electroporation cuvette.

· Set the electroporator to 1250 V, 10 µF, 600 Ohms. (This setting belongs to an Eppendorf® Electroporator 2510 using an electroporation cuvette with a gap of 1 mm. Other devices can be used, but the voltage has to be fixed at 1350V and the length of the pulse should be 5 ms.)

·Carefully knock the cuvette on the table to remove air bubbles and dry the metallic sides of the cuvette with a tissue. Do not touch the metallic sides with your hands.

·Place the cuvette into the holder of the electroporator, insert, and push the “pulse” button twice.

·Add 1 ml LB medium without antibiotics to the cuvette. Resuspend the cells carefully by pipetting up and down and pipette back into the reaction tube (avoid air bubbles in the suspension).

·Recover the culture at 37 °C in an Eppendorf thermomixer with shaking at 950 r.p.m. for 1 h 40min.

· Spin down the cells by centrifugation at 8,000g for 1 min at RT.

· Discard the supernatant by decanting, leaving about 100 ul of LB medium. Resuspend the cells by pipetting up and down.

· Streak the entire mixture with a sterile 10ul loop on one LB agar plate containing 15 ug ml⁻¹ chloramphenicol and 10ug ml⁻¹kanamycin.

Chracterize the strength of different promoters by luciferase firefly assay

preparation of the overnight p.fluorescence culture transformed successfully

Inoculate the medium in the 1.5-ml tube with the several colonies of p.fluorescence cells that has been transformed successfully and incubate at 30 °C overnight with shaking at 950 r.p.m. in an Eppendorf thermomixer.

·Puncture the cap of a 1.5-ml microcentrifuge tube, add 1.8 ml of LB containing 5ug ml−1 chloramphenicol and inoculate with 150 ul of the overnight culture.

·Incubate the mixture at 30 °C for 2h with shaking at 950 r.p.m. in an Eppendorf thermomixer.

·Adjust the mixture to OD600 0.2

Preparation of bacterial cell lysate

·Add 10ul of 1M K2HPO4(Ph7.8) and 20mM EDTA to 90ul of transformed bacteria in a new 1.5ml microcentrifuge tube.

·Quick-freeze on dry ice, and then equilibrate to room temperature by placing the tube in room temperature water.

·Add 300ul freshly prepared lysis mix (1 volume of freshly prepared lysozyme and 2volumes of 2×CCLR with 5mg/ml BSA). Mix and incubate for 10 minutes at room temperature.

· Mix 20ul of cell lysate with 100ul of luciferase assay reafent and measure the light produced.

Measrue the light produced using GloMaxTM 96 Microplate Luminometer

Colleborate with seven Chinese iGEM teams to characterize the promoter strength under differdent conditions.

modeling

Digest Pseudomonas putida S16 genome with SpeI restriction enzyme.

The purpose of this work is to achieve nicotine degradation in Pseudomonas fluorescences with the nicotine degradation gene cluster of Pseudomonas putida S16. Besides, we want to test if our promoter library could really work in regulating gene expression. The expression of nicotine degration gene cluster will be detected by the change of nicotine’s content through HPLC.

·Digest 50ug of S16 genome DNA with SpeI restriction enzyme in 400ul and incubate it at 37℃. Mix the following in a 1.5-ml microcentrifuge tube:

·Incubate the mix in 37℃ for 3h

·Add 400ul of phenol-chloroform-isoamyl alcohol mixture(25:24:1)to the 400ul digestion mixture from Step.1, mix it thoroughly by inversion to create an emulsion and centrifuge it at 9400r for 10min at RT.

·Using an end-cut wide-bore 1ml tip, gently pipette 300ul of the aqueous phase(top layer)into a new 1.5ml microcentrifuge tube.

·Add 20ul(1/15 volumes) of 3M sodium acetate(PH 7.5)and gently invert to mix.

· Add 800ul(2.5 volumes) of absolute ethanol and gently invert to mix.

· Centrifuge at 9400r for 2min at RT to pellet the genomic DNA.

·Discard the supernatant and invert the tube on a paper towl to let all of the liquid drain off the pellet.

· Wash the pellet by carefully adding 1ml of 70%(vol/vol)ethanol and centrifuge it at 9400r for 1min at RT.

·Discard the supernatant and invert the tube on a paper towl to let all of liquid drain off the pellet.

·Dry the pellet without shaking on an Eppendorf thermomixer at 45℃ for 10min until no liquid is left.

·Resuspend the DNA pellet in 12ul of autoclaved ddH2O and use 2ul to measure the concentration using the NanoDrop UV spectrophotometer. The concentration is 1ug/ul.

· Keep the DNA pellet at 4℃.