TOOLS

General Protocols

1 Stock solution

50 mg/mL Ampicillin
-0.5 g Amp, 10 mL water, filter sterilize with millipore express membrane, freeze in aliquots
50 mmol/L Arabinose
- 0.1876 g Arabinose, 25 mL water, filter sterilize with millipore express membrane.

2 Preparation of Competent Cell

Thaw an aliquot of cells (without any plasmid in them) on ice
- To 20 mL of sterile LB, add 100 μL aliquot of the thawed cells: remember, this LB does not have any antibiotic in it, so work as aseptically as possible (i.e. autoclave all solutions and use sterile pipettes).
- Grow cells in the shaker at 37 ℃ and 200 rpm, until they reach an OD₆₀₀= 0.3 - 0.4. This usually takes 1.5 - 2 hours.
- Ice down the LB with growing cells for 30 min.
- Aliquot into sterile 1.5 mL tubes and spin down at 1500 xg for 5 min at 4 ℃; discard supernatant.
- Ice down Solution A and Solution B of TaKaRa Competent Cell Preparation Kit during centrifugation.
- Gently resuspend each pellet with 100 μL Solution A.
- Centrifuge 1500 xg for 5 min and discard supernatant.
- Resuspend each pellet on ice in 100 μL Solution B.

3 Transformation

- Add 10 μL of DNA. Swirl gently with pipette.
- Incubate tubes on ice for 30 min
- Heat pulse tubes in 42 ℃ water bath for 90 s.
- Incubate on ice for 5 - 10 min
- Add 400 μL of LB broth to each tube and incubate for an hour at 37 ℃ with shaking.
- Spread 100 μL of each culture on an LB agar plate containing the appropriate antibiotics and incubate overnight at 37 ℃ (spread using beads).

4 Plasmid Purification

- Centrifuge sample in eppendorf tube approximately 1.5 mL at a time, draining off supernatant after each spin and adding more cell solution.
- Resuspend the pelleted cells in 250 μL of the resuspension Solution (mixture with Solution I and RNasa A). The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
- Add 250 μL of the Lysis Solution (Solution II) and mix thoroughly and gently by inverting the tube 5-6 times, letting it stand for 1-2 min at room temperature until the solution becomes viscous and slightly clear.
- Add 350 μL of the Neutralization Solution (Solution III) and mix immediately and thoroughly by inverting the tube 5 - 6 times.
- Centrifuge for 10 min at 12,000 rpm to pellet cell debris.
- Apply the supernatant to the supplied spin column by decanting. Avoid disturbing or applying the white precipitate.
- Centrifuge for 1 min at 12,000 rpm. Discard flow-through and place the column back into the same collection tube.
- Add 500 μL of the Wash Buffer PB to the spin column. Centrifuge for 1min at 12,000 rpm and discard flow-through. Place the column back into the same collection tube.
- Add 500 μL of the Wash Buffer W to the spin column. Centrifuge for 1min at 12,000 rpm and discard the flow-through. Place the column back into the same collection tube.
- Repeat the step 9 again.
- Discard flow-through and centrifuge for an additional 3 min to remove residual Wash Solution.
- Place the spin column in a clean 1.5 mL centrifuge tube, and pipet 20 μL Elution Buffer TE (prewarm to 60 ℃) directly to the center of the column without touching the membrane. Let it stand for 2 min at room temperature and centrifuge for 1 min at 12,000 rpm.
- Discard the column and store the purified plasmid DNA at -20 °C.

5 Standard BioBrick Assembly

- Digestion of insert: 2 - 5 μg DNA / 100 μL volume, 10× H buffer, EcoR I, Spe I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try avoiding staining or exposure to ultraviolet light of the insert.
- Digestion of vector: 2 - 5 μg DNA / 100 μL volume, 10× M buffer, EcoR I, Xba I. Digestion and inactivation. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel, try to avoid staining or exposure to ultraviolet light of the insert.

6 Suffix Insertion

- Digestion of insert: 2 μg~5 μg DNA / 100 μL volume, 10× M buffer, Xba I, Pst I. Digestion and inactivation. Clean up the insert.
- Digestion of vector : 2 μg~5 μg DNA / 100 μL volume, 10× H buffer, Spe I, Pst I. Digestion and inactivation. Clean up the vector.

7 Ligation

- After digestion and clean-up, the next step is ligation.
According to the ligation equation of Insert/Vector=3 mol/mol, combine the vector, insert, 10× T4 ligation buffer and 1 μL of T4 ligase in a 10 μL reaction overnight at 4 ℃.

8 Gel Extraction

- Weigh a 1.5 mL centrifuge tube for each DNA fragment to be isolated and record the weight.
- Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close as possible to the DNA to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weigh. Record the weight of the gel slice.
- Add Bing Buffer BD at a ratio of 100 μL of solution per 100 mg of agarose gel slices.
- Incubate the gel mixture at 55-65 ℃ for 7-10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved.
- After the dissolved gel mixture cool down, transfer it to the Spin Columns assembly and incubate for 2 min at room temperature.
- Centrifuge the Spin Columns assembly in a microcentrifuge at 12,000 rpm for 1 min, and discard the flow-through.
- Wash the columns by adding 500 μL of Wash Buffer PE to the Columns. Centrifuge the columns assembly for 1 min at 12,000 rpm, and discard the flow-through.
- Repeat step 7 again.
- Centrifuge the Columns for an additional 3 min to completely remove residual wash buffer.
- Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
- Place the spin column in a clean 1.5 mL microcentrifuge tube, and pipet 20 μL deionized water (pH is 8.0 - 8.5 and prewarm to 60 ℃)directly to the center of the column without touching the membrane. Incubate at room temperature for 2 min.
- Centrifuge for 1 min at 12,000 rpm. Discard the columns and store the microcentrifuge tube containing the eluted DNA at–20 ℃ .

9 Restriction analysis

- Combine H₂0, 4μL of dNTP Mixture, 10×Ex Taq Buffer, 0.25μL of Ex Taq, 1 μL of VR primer, 1 μL of VF2 primer and pick the colony in a 25 μL reaction for PCR.
- Gel electrophoresis.

10 Genetic mutations

1. Design the primers with mutation.
2. PCR.
- 0.25 μL of Pyrobest DNA Polymerase (5U/μL).
- 5 μL of 10 x Pyrobest buffer II.
- 4 μL of dNTP Mixture (2.5 mM each).
- 1 μL of Primer 1 (20 uM).
- 1 μL of Primer 2 (20 uM).
- 0.01 – 1 ng Template.
- Add ddH₂O up to 50 μL.
3. PCR condition.
- 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 5 min . circulate for 24 times.
4. Run the gel and do the gel extraction.
5. Blunting Kination.
- 1 pmol of DNA Fragment.
- 2 μL of 10 x Blunting Kination Buffer.
- 1 μL of Blunting Kination Enzyme Mix.
- Add ddH₂O up to 20 μL.
- Reaction under 37 ℃ for 10 min.
- Reaction under 70 ℃ for 10 min.
6. Ligation.
- Take 5 μL reacting solution into a new PCR tube.
- Add 5 μL Ligation Solution I.
- Reaction under 16 ℃ for 1 h.
- Transfor the reacting solution into 100 μL competent cell.

Characterization

Fluorescence Measurements

- The samples to be tested are cultured from plates in 20 mL of the Basal Minimal Medium with appropriate antibiotics and incubated overnight at 37 ℃ at 200 rpm.
- The culture is checked for OD₆₀₀ next day and then subculture by the same medium with antibiotics at 37 ℃ shaking for 2 h.
- Add corresponding inducer at concentration gradients into the above-mentioned culture and keep on incubating. During the time incubating, every 15 min, take 1 mL bacteria liquid, then centrifuge the cells( 6000 rpm, 10 min ) and resuspend them in 1 mL PBS. At last, pipette to a 96 well plate.
- The plate reader made by Molecular Device then read.
- The program does the following:
- In endpoint reads, following measurements are taken in a time interval of 15 min: absorbance (600 nm filter) and fluorescence (485 nm and 520 nm for GFP).
- The results then transfer to excel sheet and interpret.