Team:Shanghai HS ID/Experiments

Shanghai_HS_ID

Experiments

I. General Protocols

1. MRS media preparation

10g Tryptone
2g Meat extract
4g Yeast extract
20g Glucose
2g K2HPO4
5g Sodium acetate trihydrate
2g (NH4)2 citrate
0.2g MgSO4-7H2O
0.05g MnSO4-H2O
1g Tween-80
With up to 2L distilled water

2. LB media preparation

10g Tryptone
5g Yeast extract
10g NaCl
15g Agar
With up to 1L distilled water

3. Obtaining the Lactobacillus Casei colony

• Culture Lactobacillus. casei by using the streak plate method to get a single bacterial colony
• Incubate in 37℃ conditions overnight

4. Kanamycin solution preparation

• Combine 0.15g Kanamycin with 3mL water to prepare a 50μg/mL medium.
• Use vortex mixer to melt the Kanamycin
• Sterilize the solution with a 0.22μm aperture filter paper
• Suck up the kanamycin solution into the syringe
• Remove the needle and put it on the filter
• Squeeze the solution into a 15ml PCR tube
• The complete sterilization procedure should be done in the ultra-clean cabinet

5. Resuscitation media preparation

• MRS (liquid form)
• 50 uM glucose
• 20 uM magnesium chlorides
• 2 uM calcium chlorides

II. Construction of the recombinant plasmid

To achieve our goal, we need to construct a recombinant plasmid pNCas9-LSEI-2094 that contains the sgRNA, which guides the Cas9 protein to locate the target gene. This construction has 5 main steps and some other auxiliary experiments, as presented below.

A. Amplification of sgRNA and homologous arms,
B. Enzyme digestion that cuts the circular plasmid into a linear form
C. Connection of the plasmid and sgRNA
D. Transformation
E. Verification

1. Amplify right and left homology arms, sgRNA through PCR

•Amplification of upstream and downstream homology arms
•Design primers (around 1000 bp for each homology arm)
•Go to website NCBI to obtain the DNA sequence of L.casei ATCC 334
•Use online software to design the primers for upstream and downstream homology arms of LSEI-2094 gene
•ATCC 334 bacterium solution as a template
•334 2094-up-F/ 334 2094-up-R as primers for upstream homology arm
•334-2094-down-F / 334 2094-down-R as primers for downstream homology arm
•PrimeStar is used as the DNA polymerase

PCR protocol:

（1）95℃ 2min
（2）98℃ 10s
（3）53℃ 15s
（4）72℃ 90s
（2）-（4）repeat for 30 times
（5）72℃ 5min
（6）12℃ ∞（preserve in a low temperature）

PCR reaction system:

• Amplification of sgRNA
• Design primers
• Go to website NCBI to search for the DNA sequence of LSEI-2094 gene in L.casei ATCC 334
• Design sgRNA and its primer
• Amplify sgRNA1
• pLCNICK as a template
• Use 334 2094-sgRNA-F/ 334 2094-sgRNA-R1 as primer to amplify sgRNA1
• Amplify sgRNA2
• sgRNA1 as a template
• Use BSAI-334 2094-sgRNA-F/ BSAI-334 2094-sgRNA-R2 as primer to amplify sgRNA2

PCR protocol: same as the one for homology arm amplification
PCR reaction system:

2. Enzyme digestion to cut the plasmid into linear form

Use enzymes XbaI and ApaI for double enzyme digestion
Enzyme digestion reaction system:

The electrophoresis result of the product after double enzyme digestion

1-4 are pLCNICK after enzyme digestion, 5 is pLCNICK plasmid before enzyme digestion, 6-7 are upstream homologous arms after PCR, 8-9 are downstream homologous arms after PCR.
Use gel extraction to recycle the homologous arms and plasmids)

3. Homologous recombination

• The mass of the carrier is the length of the carrier (1200bp) times 0.02, giving a result of 240 μg. As it’s out of the available range (50-200 μg ), we instead use the minimum of 50μg
• The DNA mass we need is the length of the DNA ( 2200 bp) times 0.04, 88 μg, which can be used directly. The volume of the carrier and DNA is obtained from the formula mass/ concentration measured ( unit: μL )
• The connection system is 20 μL, which contains :
• Carrier ( 50/concentration )
• DNA ( 88/concentration )
• Exnase II enzyme ( 2 μL )
• CE II buffer ( 5* concentration ) 4μL
• Make up the rest with water
• (the steps are all done on ice)

Reaction system:

Use pipette to blend, collect the liquid after brief centrifugation

4. Transform the constructed plasmid into DH5α and add kanamycin to the LB medium to culture the DH5α

• Take out E. coli DH5α competent cells from -80 ℃ refrigerator
• Add reconstructed plasmids for every 100 μL competent cells
• Ice-bath for 30 min
• Water-bath at 42 ℃ for 45 s, and immediately place it on ice for 3 min
• Add 700 μL liquid LB medium
• Shake culture at 37 ℃ for 1 hour
• Spread coat 100μL of the mixture on the LB plate that has kanamycin (50μg/mL)
• Inverted culture at 37 ℃ for 12-16 hours

5. Extract the plasmid from the cultured DH5α

• Column balancing steps: Add 500 μL of BL buffer into the adsorption column with a collection tube, centrifuge for 1 min ( with a centrifugal force of 12000 g ), pour out the waste liquid in the collection tube.
• Add (1-5mL) of the cultured DH5α into the centrifugal tube, centrifuge for 1 min with 12000g of the centrifugal force to make the bacteria precipitate on the pipe wall, and empty the supernatant.
• Add P1 buffer (250 μL) into the centrifugal tube which contains the precipitate of bacteria, use vortex mixer to make bacteria suspend
• Add P2 buffer (250 μL) into the centrifugal tube, flip upside down gently to fully lyse the bacteria ( a vigorous shock may break the DNA )
• Add P3 buffer (350μL) into the centrifugal tube, flip upside down gently for 6-8 times. At this point, a milky precipitate may appear (the lysate of the bacteria), then centrifuge for 10 min with a centrifugal force of 12000g
• Remove the supernatant from the last step into an adsorption column (which contains a collection tube), avoid the precipitate, centrifuge for 1 min, and pour out the waste liquid in the collection tube.
• Add 600μL of PW already containing alcohol into the adsorption column, centrifuge for 1 min, pour out the waste liquid in the collection tube. (twice)
• Centrifuge for 2 min to remove the remaining buffer. Open the lid of the adsorption column and let stand for 2-3 mins at room temperature to dry the remaining buffer completely, in case the alcohol may affect the later experiment.
• Put a new EP tube below the absorption, aim at the adsorption column membrane to add the EB buffer (100μL), let stand for 2 mins at room temperature/ centrifuge for 2 mins to make the plasmid enter the EP tube.
• Testify the plasmids
• Pick multiple colonies and add them into 10μL LB medium. Use it as a template for PCR. Use 334 2094-down-F/ 334 2094-down-R as primers testify through PCR
• Electrophoresis result:

• Add the colonies that are tested positive into 3 mL liquid LB medium that has 50 mg/L kanamycin, 200rpm, and shake the culture at 37℃

6. Test whether the reconstruction of the plasmid is successful

• Deliver the plasmids to Tsingke Biotechnology for DNA sequencing
• Primer: Nickcas-seq-F/Nickcas-seq-R
• Compare and analyze the result according to the sequencing result on software Snapgene

III. Obtainment of the modified L. casei strains

After the construction of the plasmid, we need to get the modified L.casei strains by transforming the plasmids into more L.casei, which needs electroporation.

1. Preparation of competent cell for L.casei（all procedure should be done on ice）

• Culture the single colony of L.casei in the constant temperature shaker until its value of OD600 reaches approximately 0.6 (at which the bacteria has reached the plateau developmental phase)
• Pour the bacterium solution into centrifugal tubes of 50mL (pre-cooled), centrifugate (4℃, 5000rpm, 10min)
• Pour out the supernatant, add 100mL of double-distilled water (pre-cooled) and mix properly to let bacteria re-suspense (avoid using the vortex mixer because huge mechanical force might break the bacteria). Repeat this step.
• Pour out the water, add 2mL of water (pre-cooled), mix it to re-suspense, then distribute the bacterium solution into a centrifugal tube of 1.5mL, centrifugate (4℃, 13000rpm, 3min), pour out the water.
• Add 1mL of water in each tube, mix it gently, centrifugate (4℃, 13000rpm, 3min), pour out the water, do this step twice to ensure that there are no impurities
• Add 500μL of PEG-8000 (30% concentration), re-suspense, centrifugate (4℃, 13000rpm, 3min)
• Add 250μL of PEG-8000 (pre-cooled) each tube, re-suspense, then distribute to pre-cooled centrifugal tubes (100μL per tube)

2. Electroporation

• Unfreeze the L.casei competent cells and the constructed plasmids at room temperature
• Add 150 mL H2O into competent cells and place the mixture at room temperature for 30 minutes
• Centrifugate the mixture (4 Celsius, 13000rpm, 3 minutes)
• Add 500 uL PEG-8000 and centrifugate the mixture again (4 Celsius 13000rpm, 3 minutes)
• Add 100 uL PEG-8000
• Add 10 uL plasmid and do electroporation
• Electric capacity: 25 uV
• Voltage: 2000V
• Resistance: 400 ohms
• Add 900 uL resuscitation medium and shake it for 3 to 4 hours
• Take 100 µL mixture and spread coat it on solid MRS medium, culture it in the incubator at 37 ℃ for 3-4 days, observe its growth

3. Use PCR to testify

• Pick a colony of L.casei and add it into 10 µL of MRS medium, use 1 µL as template for PCR

• Conduct electrophoresis

4. DNA sequencing

• Deliver the plasmids to Tsingke Biotechnology for DNA sequencing
• Primer:2094-seq-F / 2094-seq-R
• Compare and analyze the result according to the sequencing result through NCBI/BLAST

IV. Result

Compare the DNA transformation rate of wild type L.casei and L.casei with LSEI-2094 gene being knocked out

1. Prepare ATCC 334 competent cells for wild type and LSEI-2094 defect type
2. Electroporation: add plasmids (10 µL plasmid for 100 µL bacterium solution)
3. Cultivate L.casei with resistance cell culture

• Place the bacteria into the incubator at 37 ℃ For 3-4 hours
• Use a pipette to draw two types of competent cells and spread coat them on MRS medium with 3 µg/mL erythromycin
• Cultivate them for 4 days