Team:SCU China/Notebook

iGEM SCU experiment protocol

Transformation

1. Thaw the competent cells on ice for about 10-15 minutes.
2. Add 50 μL of thawed competent cells into each pre-chilled 1.5mL eppendorf tube.
3. Add 1.5 μL of the resuspended DNA (or 4 μL of the ligation product) to the 1.5 mL tube, gently pipet up and down for a few times, make sure to keep the competent cells on ice.
4. Close the tubes and incubate the cells on ice for 30 minutes.
5. Heat shock the cells by immersion in a pre-heated water bath at 42 ℃ for 60-90 seconds.
6. Incubate the cells on ice for 5 minutes.
7. Add 300 μL of SOC (make sure that the broth does not contain antibiotics and is not contaminated) to each tube.
8. Incubate the cells at 37 ℃ for 1-2 hours while the tubes are rotating or shaking (The purpose of this step is to ensure the transformation efficiency).
9. Distribute 200 μL transformed cells uniformly over the appropriate LB plate (containing the proper antibiotics or not).
10. Incubate the plates at 37 ℃ for 12-14 hours, make sure the agar side of the plate is up.

Liquid culture to grow bacteria from a single colony

1. Add 5 mL LB into each 10 mL or 15 mL tube.
2. Add proper antibiotics solution into each tube；
3. Pick a single colony on plates with tweezers clamping a pipette tip and then drop the tip containing transformed cells into appropriate LB containing proper antibiotics.
4. Seal tubes with sterile sealing film.
5. Incubate the cells at 37 ℃ for overnight while the tubes are rotating or shaking.

3A  Assembly

1. Restriction Digests

1. The left part sample is cut out with EcoR I and Spe I.
2. The right part sample is cut out with Xba I and Pst I.
3. The linearized plasmid backbone, which is harvested by PCR，is a linear piece of DNA. It has a few bases beyond the EcoR I and Pst I restriction sites. It is cut with EcoR I and Pst I.

1. All 3 restriction digests are heated at 80 ℃ for 20 minutes in PCR meter to heat kill all of the restriction enzymes.
2. An equimolar quantity (relative quantity) of all 3 restriction digest products are combined in a ligation reaction.
3. The desired result is the left part sample's Spe I overhang ligated with the right part sample's Xba I overhang resulting in a scar that cannot be cut with any of our enzymes.
4. The new composite part sample is ligated into the construction plasmid backbone at the EcoR I and Pst I sites.
5. When the ligation is transformed into cells and grown on plates with antibiotic C, only colonies with the correct construction are survived.

Traditional assembly (standard assembly) methods

For Standard Assembly, a part sample is cut out from its plasmid backbone and inserted into the prefix of a plasmid backbone of another part (we use EcoRⅠand Xba Ӏ to digest plasmid backbone and use EcoRⅠand SpeⅠto digest the target part). Two restriction digests are done, one for the part sample that will be moved and one for the plasmid backbone that will receive it. The digests are then run on a gel and using gel purification the required fragments are isolated (the part sample and the cut plasmid backbone). The purified insert and cut plasmid backbone are ligated and the resulting composite part can be transformed into E. coli cells.

Miniprep protocol

   We use MINIPREP PLASMID KIT (TIANGEN BIOTECH, CHINA) for miniprep.

Restriction digestion

   We conduct our experiments following the standard protocol from the HD, except for no adding BSA into our mixture.

Ligation

   We follow the standard protocol from HD web to conduct our ligation experiments.

Making linearized plasmid backbones

   We do this by the bulk production protocol from HD.

Competent cells

   We choose DH5α from TIANGEN BIOTECH as our competent cells meterials.

Quantifying fluorescence of GFP expressed in E. coli

We choose plate reader to detect fluorescence of GFP expressed in E. coli.

Before detecting the fluorescence on plate reader, we view the fluorescence of E. coli with a fluorescence microscope to make sure that GPF expresses normally in vivo.

Incubate bacteria for certain periods of time, then take some sample out and add it into each well on 96 well plate (for our experiment, we add 150 μL per well).