1.LB-Media

LB Liquid Medium（1L）

2.Transformation

1. Thaw competent cells on wet ice. Place the required number of 1.5mL eppendorfs on wet ice, then make 100 µL aliquots of competent cells in the chilled 1.5mL microcentrifuge tubes.

2. Add 10 µL of sample DNA directly into a tube of competent cells. Mix well by gently flicking tube several times.

3. Incubate the cells on ice for 30 minutes.

4. Heat-shock the cells for exactly 30 seconds in a 42 ℃ water bath.

5. Incubate the cells on ice for 2 minutes.

6. Add 900 µL of LB Liquid Medium.

7. Shake the tube at 200 rpm for 1 hour at 37 ℃.

8. Centrifugate eppendorfs at 12000rpm for 3 minutes and discard 900μL supernatant. Resuspend cells with the remaining liquid.

9. Spread plates with bacterial suspension and incubate for the night.

3.Plasmid Extraction

(According to FastPure^® Plasmid Mini Kit)

1. Take 1-5 mL bacterial solution into a centrifuge tube, centrifuge at 12,000 rpm for 1 min and remove supernatant.

2. Add 250 μL SolutionⅠ in centrifuge tube, using the pipet or vortex oscillator to suspend the cells.

3. Add 250 μL Solution II in centrifuge tube and gently flip upside down for 6-8 times to make sure the germ is full cracked.

4. Add 350 μL Solution III, gently flip upside down for 6-8 times to mix until white, flocculent precipitate appears and centrifuge at 12,000 rpm for 10 minutes.

5. Add the supernatant to the adsorption column in step 5, centrifuge at 12,000 rpm for 1 minute, discard the filtrate and reuse collection tube.

6. Add 700 μL Wash solution to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the filtrate and reuse collection tube.

7. Add 500 μL Wash solution to the adsorption column, centrifuge at 12,000 rpm for 1 minute, discard the filtrate and reuse collection tube.

8. Centrifuge the empty adsorption column at 12,000 rpm for 2 minute to dry the column matrix. (Residual ethanol may impact downstream application)

9. Transfer the adsorption column into a clean 1.5 mL centrifuge tube, add 50 -100 μL Elution buffer to the center of the column membrane, let sit at room temperature for 2 minutes and centrifuge at 12,000 rpm for 1 minute, collect the plasmid solution in the centrifuge tube.

10. Store the plasmid at -20 ℃.

4.Ligation

Components(10μL)	Volume/μL
T4 DNA ligase	1
10 × T4 DNA Ligase Buffer	1
Plasmid Skeleton	molar ratio of Vector: plasmid is 1:3
Insert Gene	molar ratio of Vector: plasmid is 1:3
ddH2O	Up to 10 μL

Overnight at 16 ℃.

5.Electrophoresis

1. Place the gel tray in the appropriate position in the gel cartridge and place the comb in the correct position.

2. Measure 0.5 g agarose, put it in a 250 mL Erlenmeyer flask, add 50 mL 1 × TAE buffer and mix, then put the Erlenmeyer flask in the oven and heat to boil until the agarose is completely dissolved.

3. Add 5 μL GelRed to the solution.

4. Pour the solution into the gel casting tray.

5. After the gel cools to solid, pull out the comb.

6. Place the gel in the electrophoresis chamber with enough TAE buffer.

7. Add 10 × loading buffer to the sample and mix, then transfer the mixture to the well on the gel with a pipette.

8. Power on, run at 120 V for half an hour.

6.Gel Extraction

(According to FastPure^® Gel DNA Extraction Mini Kit)

1. Perform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments. Any type or grade of agarose may be used. However, it is strongly recommended that fresh TAE buffer or TBE buffer be used as running buffer. Do not reuse running buffer as its pH will increase and reduce yields.

2. When adequate separation of bands has occurred, carefully excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel slice by removing extra agarose.

3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5 mL microcentrifuge tube. Assuming a density of 1 g/mL, the volume of gel is derived as follows: a gel slice of mass 0.3 g will have a volume of 0.3 mL.

4. Add 1 volume Binding Buffer (XP2).

5. Incubate at 50-60 ℃ for 7 minutes or until the gel has completely melted. Vortex or shake the tube every 2-3 minutes.

6. Insert a HiBind^® DNA Mini Column in a 2 mL Collection Tube.

7. Add no more than 700 μL DNA/agarose solution from Step 5 to the HiBind^® DNA Mini Column. Centrifuge at 10,000 xg for 1 minute at room temperature. Discard the ltrate and reuse collection tube.

8. Repeat Steps 7 until all of the sample has been transferred to the column.

9. Add 300 μL Binding Buffer (XP2). Centrifuge at maximum speed (≥13,000 xg) for 1 minute at room temperature. Discard the ltrate and reuse collection tube.

10. Add 700 μL SPW Wash Buffer. Centrifuge at maximum speed for 1 minute at room temperature. Discard the ltrate and reuse collection tube.

11. Centrifuge the empty HiBind^® DNA Mini Column for 2 minutes at maximum speed to dry the column matrix. Transfer the HiBind^® DNA Mini Column to a clean 1.5 mL microcentrifuge tube.

12. Add 50 μL deionized water directly to the center of the column membrane. Centrifuge at maximum speed for 1 minute.

13. Store DNA at -20 ℃.

7. PCR

1. Prepare the reaction system in a PCR tube on ice, thaw the components and mix well. After use, put them back in -20 ℃.

2. Gently centrifuge to collect the liquid at the bottom of the tube.

3. Transfer the PCR tube to the PCR machine, set the parameters and start the thermal cycle.

8.HR

(According to ClonExpress^® Ultra One Step Cloning Kit)

1. Obtain linear vectors and inserts by PCR.

2. Purify PCR products, and then detect concentration.

3. The optimum cloning vector usage and insert fragment usage for ClonExpress® II recombination reaction system is 0.03 pmol and 0.06 pmol, respectively. According to this introduction, the corresponding DNA quality can be roughly calculated by following formulas.

① Optimal cloning vectors usage= [0.02* Base pairs number of cloning vectors] ng;

② Optimal inserts usage= [0.04* Base pairs number of inserts] ng.

③ Note: Linear cloning vectors should be used between 50 and 200 ng; insert amplification products should be used between 10 and 200 ng. Select the lowest/highest amount directly when the optimal amount of DNA used exceeds this range by using the above formula.

4. Arrange the following reaction systems on ice.

9.SDS-PAGE

1. Wash glass plates and spacers, place rubber seal in bottom of gel kit and put the spacers in between the two pieces of glass, making sure they are level.

2. Add the clamps and plugs, dispense 12.5% separation gel. Shake well immediately after adding TEMED to fill the gel. When filling the gel, 5 mL of gel can be sucked out along the glass by the gun, and the gel surface can be raised to a height of 1 cm under the rubber comb. Then add a layer of ethanol to the gel, and the gelation after liquid sealing is faster.

3. Placed at room temperature for 30 min to be solidified, when there is a line of refraction between ethanol and gel, the gel has been condensed. When the gel is fully solidified, the upper layer of ethanol can be poured off and the ethanol is blotted dry with absorbent paper.

4. Dispense 5% concentrated gel. Immediately after adding TEMED, the mixture can be filled. Fill the remaining space with the concentrated gel and insert the comb into the concentrate. When filling the gel, the gel should also flow down along the glass plate to avoid bubbles in the gel. Keep the comb level when inserting the comb. Since the volume shrinks and shrinks when the gel solidifies, the loading volume of the sample hole is reduced, so the gel is often applied on both sides during the solidification process of the concentrated gel. After the gel has solidified, pinch the sides of the comb and pull them straight up.

5. Make up running buffer (1 in 10 dilution of stock). Need around a liter.

6. Remove comb and place gel in opposite side of apparatus.

7. Load samples and molecular weight markers. 15 μL of protein from supernatant and 5 μL marker.

8. Put on grey plastic top (make sure rubber seal is in place).

9. Add the plugs and slowly add running buffer.

10. Put on front plastic cover and run at 120V for 1 hour.

10.Purification

(According to High Affinity Ni-NTA Resin)

1.Inoculate recombinant E. coli BL21 in 20 mL of LB liquid medium containing 50μg/mL kanamycin, and cultivate them overnight at 37℃ with shaking at 180 rpm. Inoculate 2% of the overnight cultured bacteria in 100 mL of LB liquid medium containing 50μg/mL kanamycin, and culture them with shaking at 25℃ for 24 hours.

2.The bacterial cultures were harvested and centrifuged at 12000 rpm for 10 min at 4℃.

3.For each culture supernatant, 300 mL of them was concentrated using MilliporeSigmaTM AmiconTM Ultra Centrifugal Filter Units (3 kDa) to about 10 mL in total.

4.Wash the Ni-NTA column with 50 mL PBS.

5.Load the concentrated sample in the Ni-NTA column for several times.

6.Wash the Ni-NTA column with 50 mL PBS.

7.Elute the Ni-NTA column with 200 mM 1.5mL 250 mM imidazole.

8.Wash the Ni-NTA column with 50 mL PBS and store it at 4℃ by adding 10 mL ethanol.

9.Store the purified protein at -20℃.

11.Running the SDS-PAGE Gel

1.Wash glass plates and spacers, place rubber seal in bottom of gel kit and put the spacers in between the two pieces of glass, making sure they are level.

2.Add the clamps and plugs, dispense 12.5% separation gel. Shake well immediately after adding TEMED to fill the gel. When filling the gel, 5 mL of gel can be sucked out along the glass by the gun, and the gel surface can be raised to a height of 1 cm under the rubber comb. Then add a layer of ethanol to the gel, and the gelation after liquid sealing is faster.

3.Placed at room temperature for 30 min to be solidified, when there is a line of refraction between ethanol and gel, the gel has been condensed. When the gel is fully solidified, the upper layer of ethanol can be poured off and the ethanol is blotted dry with absorbent paper.

4.Dispense 5% concentrated gel. Immediately after adding TEMED, the mixture can be filled. Fill the remaining space with the concentrated gel and insert the comb into the concentrate. When filling the gel, the gel should also flow down along the glass plate to avoid bubbles in the gel. Keep the comb level when inserting the comb. Since the volume shrinks and shrinks when the gel solidifies, the loading volume of the sample hole is reduced, so the gel is often applied on both sides during the solidification process of the concentrated gel. After the gel has solidified, pinch the sides of the comb and pull them straight up.

5.Make up running buffer (1 in 10 dilution of stock). Need around a liter.

6.Remove comb and place gel in opposite side of apparatus.

7.Load samples and molecular weight markers. 15 μL of protein with SDS-PAGE sample loading buffer(4X) from supernatant and 5 μL marker.

8.Put on grey plastic top (make sure rubber seal is in place).

9.Add the plugs and slowly add running buffer.

10.Put on front plastic cover and run at 120V for 1 hour.

12.The Effect of Temperature

This biobrick is presented in plasmid pET-29a(+), which harbours a Kanamycin resistant gene as a selection marker. So the Luria-Bertani (LB) plate and medium we used were supplemented with Kanamycin (50 μg/mL).

We chose BBa_K592101 from the iGEM distribution kit and used pET-29a(+) as the vector, which is capable of expressing LacI to conduct BBa_K4164018.Then the bacteria was coated on the LB plate, cultured overnight at 37 ℃. The next day we picked 4 colonies and incubated them in 5 mL LB medium for about 12 hours. The plasmids were extracted and transformed into E. coli BL21 and cultured overnight in 5 mL LB medium.

The overnight culture of E. coli BL21 and E. coli DH5α were diluted with fresh 150 mL LB medium and incubated at 37℃ to a 0.6 optical density at 600 nm (OD600), then separated into 5 groups, corresponding to the three temperature gradients 4,16,37,42,55℃.

The bacteria medium were being incubating with shaking at 200 rpm for 4 hours then taking a sample 50 mL which was being centrifuging at 8000 rpm for 10 minutes ,ultrasonically shattering for 15 minutes and then centrifuging at 8000 rpm for 10 minutes once again. After that sample after 30 minutes and measure the OD600 and fluorescence (excitation 484nm; emission 534nm) data using 96-well microplate reader. LB media was served as control.

13.The Effect of different concentration of IPTG

Transform the plasmids into BL21 strain.Pick up a single colony by a sterile tip from each of the plates for all the experimental and control groups. And put the colony into 5mL LB medium with 50 µg/mL Kanamycin. Incubate at 37℃ in a shaker for 20h.

Detect OD600 value of the culture medium with spectrophotometer.

Add 100 µL bacteria culture medium into a sterile 96-well plate. IPTG is added to final concentrations of 0, 0.1, 0.5, 1, 5, 10mM. LB medium is the blank control. The colony without YFP expression is the negative control.

Incubate at 37℃, and measure the fluorometric value(excitation 484nm; emission 534nm) and OD600 value for each well every 1h or 2h, using an automatic microplate reader.

The experiment should be repeated at least 3 times.

Reference

Daubner, S. Colette, et al. "Yellow light emission of Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow fluorescent protein." Proceedings of the National Academy of Sciences 84.24 (1987): 8912-8916.

Parmentier, G., and H. Eyssen. "Synthesis of the Specific Monosulfates of Cholic Acid." Steroids 26.6 (1975): 721-29. Web.