Strains

E.coli DH5α

F ^- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(r_K –m_K+), λ–

• Used for HiFi assembly and amplification of plasmids
• Used for qPCR experiment in verification of ser integrase

E.coli BL21(DE3)
E. coli str. B F ^- ompT gal dcm lon hsdS_B(r_B–m_B–) λ(DE3 [lacI lacUV5-T7p07 ind1 sam7 nin5]) [malB⁺]_K-12(λ^S)

• Used for protein expression

E.coli Mach1-T1
str. W ΔrecA1398 endA1 fhuA Φ80Δ(lac)M15 Δ(lac)X74 hsdR(r_K–m_K+)

• Used for protein expression when lacZ is the reporter gene

Plasmids

pET28a (+)
pET28a(+) is a bacterial cloning vector backbone. The original pET28a (+) plasmid was kanamycin resistance, while we created another version with chloramphenicol resistance. The original pET28a(+) plasmid was a gift from our instructor Peng Li.

pBad24
pBad24 is a bacterial cloning vector backbone. The plasmid was bought from http://www.miaolingbio.com/plasmid/P0082.html

pFB48
pFB48 is a bacterial expression vector used as the source of T7 core-T3 sigma amplifier system. It was a gift from Fang Ba, ShanghaiTech University.

pFB20
pFB20 is a bacterial expression vector with a strong promoter(J23110). We used this vector to initiate expression of nisK and nisR. The device confers Chloramphenicol resistance. pFB20 was a gift from Fang Ba, ShanghaiTech University.

NEU-pmrCAB-EGFP
NEU-pmrCAB-EGFP was uesd as the reference of our molecular cloning. It has ampicillin resistance gene. It was a gift from NEU 2020 iGEM team.

Molecular Experiments

PCR
We use the Q5 High-Fidelity 2X Master Mix from BioLabs to do our PCR.

Colony PCR
We select one colony into 10 μL of water as PCR template. Then we use the Taq 2X mix to do colony PCR. The other steps are the same as normal PCR.

Agarose Gel Electrophoresis
We use 1x TAE solution to prepare the agarose gel and the electrophoresis running buffer. The concentration of agarose varies according to our propose.

Gel Extraction
We use MN PCR clean-up and gel extraction kit.

HiFi DNA Assembly
We use NEBuilder® HiFi DNA Assembly Master Mix. Before use, thaw and vortex master mix thoroughly and keep on ice.

1. Set up the following reaction on ice:



	2~3 Fragment Assembly	4~6 Fragments Assembly
Recommended DNA Ratio	vector:insert=1:2	vector:insert=1:1
Total Amount of Fragments	0.2pmol X μl	0.5pmol X μl
Master Mix	10 μl	10 μl
ddH2O	10~X μl	10~X μl
Total	20 μl	20 μl



2. Incubate samples in a thermocycler at 50℃ for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Follwing incubation, store samples on ice or at -20℃ for subsequent transformation.

Restriction Enzyme Digestion

Stop reaction by adding 10μl of 6X gel loading dye to 50μl reaction.

Biochemistry Experiments

Protein Expression

1. Prepare 1L LB media. Autoclave for 45 min and cool to room temperature.
2. Incubate each 1L LB with 10ml of the pre-culture after adding 1ml antibiotic. Shake at 37℃.
3. After 3h, check OD at 600nm. If OD=1, decrease temperature to 16℃. After 30min-1h, add IPTG for a final concentration of 0.5mM to induce plasmid expression. Shake at 16℃ for 16-20h.
4. Harvest cells at 4,000 rpm for 20 min at 4℃. Transfer pellet to a 50ml tube by PBS and centrifuge again, remove supernatant and store pellet at -80℃.

Protein Purification

1. Prepare Lysis buffer, 30ml per 1L culture.
2. Resuspend cell pellet in Denatured Lysis buffer and keep on ice.
3. Prepare ultracentrifuge: cool down to 4°C.
4. Sonicate on ice, at 180W 10sec on & 10sec off. Repeat for a total time of 20 min.
5. Spin down in ultracentrifuge: 40min at 4°C at 12000g.
6. Equilibrate Ni-RTA resin: add 10 mL 50% slurry to glass golumn, wash the resin with 25 mL wash buffer.
7. Add the supernatant to the equilibrated Ni-resin and collect the flow through. Add the flow through to the Ni-resin again.
8. Wash the column with wash buffer. Wash with at least 50ml per column.
9. Elute using 25ml elution buffer (300mM Imidazole). Collect in centrifuge tube.
10. Determine protein concentration by measuring the absorbance, blank = elution buffer, using nanodrop at A280. 1Abs=1mg/ml.
    Practical Concentration (mM)= Abs/ Ext. coefficient*1000
11. Concentrate sample to a proper concentration using Amicon ultracentrifuge filter units by spinning down at 4,000rcf at 4°C until desired volume is reached. I will make sure the final concentration can be diluted at least 50 times to reach the work concentration.
12. Transfer concentrated sample to a new tube and measure absorbance again.
13. Make aliquots: 50ml. Store aliquots at -80°C after snap-freezing in liquid nitrogen.
14. Prepare 1:100 diluted sample as SDS-PAGE samples – add 4x loading buffer and boil before either running the gel immediately or storing at -20°C.

Western Blot

1. The procedures before getting PAGE gel are the same as SDS-PAGE.
2. Blotting
• Prepare 2 copies of filter paper and one NC membrane in the same size as the gel. Soak the filter papers in transfer solution.
• Carefully place the gel, filter papers, membrane into Semi-Dry Electrophoretic Transfer Cell in the order shown in the following figure. Pay attention to drive away bubbles between each layers using 15ml centrifugal tube.



Schematic figure of Western blot.


• Transfer the membrane at 25 V, for 35 min.
• Wash the membrane with TBST for 3-5 s.
3. Blocking
• Soak the membrane in 5% milk for over 1 h at RT or at 4℃ overnight.
• Wash the membrane with TBST for 10 s. Shake.
• Wash the membrane with TBST for 5 min, twice.
4. 1st Ab incubation
• Cover the membrane with 1st Ab (1:5000) in the plate and shake for 1 h at RT.
• Wash the membrane with TBST for 10 s, shaking.
• Wash the membrane with TBST for 5 min, twice.
5. 2nd Ab incubation
• Cover the membrane with 2nd Ab (1:10000) in the plate and shake for 1 h at RT.
• Wash the membrane with TBST for 10 s, shaking.
• Wash the membrane with TBST for 5 min, twice.
6. Color reaction
• Prepare substrate solution: 200ul Enhanced Oxidizing Reagent & 200 ul Enhanced Luminol Reagent.
• Incubate membrane in substrate solution
• Use developer to take photos.

Microbiological Experiments

Transformation

1. Melt 100μL of E.coli component cells on ice.
2. Add some plasmids or HiFi products to melted 100 μL of E.coli component cells. For plasmids, 200 ng is enough; as for HiFi products, 10 μL or 20 μL is appropriate.
3. Put the mixture on ice for 15 minutes.
4. Incubate the mixture at 42 ℃ in a thermostat water bath cauldron for 90 seconds.
5. Put the mixture on ice for another five minutes, and add 1 mL of liquid LB, incubate in a table (37 ℃, 200 rpm) for 45-60 minutes.
6. Centrifuge the cultivated cells at 6000 rpm for 5 minutes, and pour out most of the supernatant. Re-suspend the cells in about 100-200 μL of supernatant, and spread on a LB plate with appropriate antibiotics added.

Plasmid Extraction

1. Plasmid mini kit
We use the TIANprep Mini Plasmid Kit bought from Tiangen Biotech co., ltd.

• Materials:     ○ 1.5 mL Eppendorf tubes
      ○ Centrifuge
      ○ Kit Buffer P1
      ○ Kit Buffer P2
      ○ Kit Buffer P3
      ○ Kit Buffer PWKit Buffer BL
      ○ Kit Spin Columns CP3
      ○ Kit Collection Tubes 2 mL
      ○ ddH2O
  
• Protocol

0) Balance the spin column

• Add 500 μL of buffer BL to the spin column CP3, put the column in a collection tube
• Centrifuge at 12,000 rpm, 1 min
• Pour out the liquids in the tube

1) Resuspend cells, lyse and neutralize:

• Centrifuge the cultivated cells (4 μL) at 12,000 rpm for 2 min
• Add to the pelleted cells:
    ○ 250 μL of buffer P1 and vortex
    ○ 250 μL of buffer P2 and gently invert the tube 6-8 times
    ○ 350 μL of buffer P3 and gently invert the tube 6-8 times at once

• Centrifuge at 12,000 rpm for 10 min

2) Bind DNA:

• Transfer the supernatant to spin column CP3
• Centrifuge for 30-60 s at 12,000 rpm

3) Wash the column:


• Add 600 μL of buffer PW and centrifuge for 30-60 s at 12,000 rpm
• Discard the flow-through
• Repeat twice
• Centrifuge empty column for 2 min at 12,000 rpm to get rid of buffer PW
• Open the lid of the column and place it at room temperature for 2-5 min

4) Elute purified DNA:


• Transfer the column into a new Eppendorf tube
• Add 30-50 μL of ddH2O (incubated at 70 ℃) to the column and incubate at 70 ℃ for 2 minutes
• Centrifuge 2 minutes at 12,000 rpm
• Collect the flow-through

5) Quantity of DNA recovered (with a Nanodrop)


2. Plasmid Maxi kit

We use the EndoFree Maxi Plasmid Kit bought from Tiangen Biotech co., ltd.


• Materials:     ○ 50 mL Eppendorf tubes
      ○ Centrifuge
      ○ Kit Buffer P2
      ○ Kit Buffer P4
      ○ Kit Buffer PW
      ○ Kit Buffer BL
      ○ Kit Filtration CS1
      ○ Kit Spin Columns CP5
      ○ Kit Collection Tubes 50 mL
      ○ ddH2O
  
• Protocol

0) Balance the spin column


• Add 2.5 mL of buffer BL to the spin column CP6, put the column in a collection tube
• Centrifuge at 8,000 rpm, 2 min
• Pour out the liquids in the tube

1) Resuspend cells, lyse and neutralize:


• Centrifuge the cultivated cells (100 mL) at 8,000 rpm for 3 min
• Add to the pelleted cells:
• 8mL of buffer P1 and vortex
• 8mL of buffer P2 and gently invert the tube 6-8 times
Place the tube at room temperature for 5 min

• 8mL of buffer P4 and gently invert the tube 6-8 times at once
• Place the tube at room temperature for 10 min
• Centrifuge at 8,000 rpm for 5-10 min
• Pour all the solution carefully to the filtration CS1, slowly push the handle, collect the filtrate in a new clean tube
• Add 0.3 times of the filtrate volume of isopropanol to the filtrate, invert the tube to mix them together and transfer the supernatant to spin column CP6

2) Bind DNA:


• Centrifuge for 2 min at 8,000 rpm
  

3) Wash the column:


• Add 10 mL of buffer PW and centrifuge for 2 min at 8,000 rpm
• squareard the flow-through
• Repeat twice
• Add 3 mL of water-free ethanol and centrifuge for 2 min at 8,000 rpm
• Centrifuge empty column for 5 min at 8,000 rpm to get rid of buffer PW
• Open the lid of the column and place it at room temperature for 2-5 min

4) Elute purified DNA:


• Transfer the column into a new 50 mL Eppendorf tube
• Add 1-2 mL of ddH2O to the column and incubate at room temperature for 5 minutes
• Centrifuge 2 minutes at 8,000 rpm
• Collect the flow-through

5) Quantity of DNA recovered (with a Nanodrop)

Verification of cro and cI via fluorescence detection

1. Expression
• Prepare 4 15 mL test tubes with 3.6 mL LB with chloramphenicol
• Add 0.4 mL of BL21 in the tubes. The first tube contains negative
  control (bacteria with the empty vector), the second one contains
  positive control (bacteria constitutively express mcherry), the rest ones contains bacteria with the verification plasmid of cro and cI
• Induce the second tube with a final concentration of 1 % arabinose for verification of cro (the third tube is used for verification of cI)
• Shake for about 3 hours at 37 ℃ until OD600 is about 0.4-0.6
• Shake for another 16 hours at 37 ℃
2. Fluorescence detection
3. Centrifuge the bacteria at 6,000 rpm for 5 minutes
4. Pour out the supernatant and re-suspend the pellet in PBS whose volume is 20 % of the original cultivate
5. Add the re-suspended liquids in an ELISA plate
6. Check fluorescence
mcherry: 552 nm excitation and 610 nm emission

7. Check OD600
8. Export two sheets as Excel
9. Calculate fluorescence intensity (mcherry/OD)
10. Compare the fluorescence intensity of four tubes
    

Verification of ser intergase via fluorescence detection

1. Expression
• Prepare 2 15 mL test tubes with 3.6 mL LB with chloramphenicol
• Add 0.4 mL of BL21 in the tubes with plasmid for verification of ser integrase.
• Shake for about 3 hours at 37 ℃ until OD600 is about 0.4-0.6
• Induce the fourth tube with a final concentration of 1 % arabinose for verification of integrase (the first tube is used as control)
• Shake for another 16 hours at 37 ℃
2. Fluorescence detection
3. Centrifuge the bacteria at 6,000 rpm for 5 minutes
4. Pour out the supernatant and re-suspend the pellet in PBS whose volume is 20 % of the original cultivate
5. Add the re-suspended liquids in an ELISA plate
6. Check fluorescence
mcherry: 552 nm excitation and 610 nm emission

7. Check OD600
8. Export two sheets as Excel
9. Calculate fluorescence intensity (mcherry/OD)
10. Compare the fluorescence intensity of two tubes

Expression of pNisin in Mach1-T1 and Fluorescence Detection

1. Expression
• Prepare a 250ml conical flask with 100ml LB+Chl
• Add 1ml Mach1-T1(V37) in the conical flask
• Shake for 5-6h until OD600nm=0.6-0.8
• Prepare 18 cell culture tubes, divede into 3 groups
(1:nisin; 2:nisA-affi elution; 3:elution buffer(300mM imidazole))

• Add 4ml pre-culture to each new tube
• Add gradient nisin(or nisAaffi, 300mM imidazole) to 3 groups of tube

Nisin(ng)	nisAaffi elution(μl)	300mM imidazole（μl）
0	0	0
0.5(0.2ng/μl*2.5μl)	0.5	0.5
1(2ng/μl*0.5μl)	1	1
5(2ng/μl*2.5μl)	5	5
10(20ng/μl*0.5μl)	10	10
100(200ng/μl*0.5μl)	100	100



• Shake for 4h in 37℃
• Transfer 2ml pre-culture to 1.5ml EP tubes(keep shaking the remaining 2ml pre-culture)
• Harvest cells at 8,000 rpm for 5 min
• Resuspension by 1ml PBS and centrifuge again
• Discard supernatant as completely as possible
• Resuspension by 500μl PBS and transfer the bacteria liquid to 3 wells of 96-well plate, 150μl per well
• Detect the flurescence by microplate reader
• When the remaining 2ml pre-culture has been shaked for another 4 h, repeat the step 9) through step 14)
2. Fluorescence detection
3. Check fluorescence
4. EGFP:488nm excitation and 513nm emission
5. Check OD600nm
6. Export two sheets as Excel
7. Calculate fluorescence intensity(EGFP)/OD

Induction of affipmrABC system

1. Cultivate the E.coli containing affipmrABC system in LB medium until the OD value reaches between 0.6 and 0.8.
2. Add IPTG to induce. The final concentration of IPTG is 0.24 mg/mL.
3. Add antibody induction to make the final concentration of antibody 100 mM.
4. Observe the fluorescence of bacteria with fluorescence microscope.