Experiments | Shanghai_city

The growth of bacteria requires carbon(c), nitrogen(n), oxygen(O), hydrogen(H), and we also needed to keep the osmosis at a balance, thus adding NaCl.

What we needed was 100mL of solid medium and 350mL of liquid medium. According to the the formula, every 1L of medium requires 10g of tryptone which provides N, 5g of yeast extract which provides C, 10g of NaCl, while O and H are from water. For the solid medium, a 1% concentration of agarose powder is needed for solidification.

After adding the ingredients together, except the agarose, we used a plastic sheet with filter paper in the middle to cover the flask and filter possible impurities during autoclaved sterilization. We sterilized for about 20 minutes and let the medium cool down. For the solid medium, we added 50µL of Kana antibiotic to kill the bacteria that didn’t contain the resistance gene. And for the final step, we poured the melted solid medium into petri dish in the clean bench.

1. PCR
2. Plasmid extraction
3. PCR Gel Electrophoresis
4. PCR Gel Purification
5. Gibson Enzyme Connection
6. DNA transformation to plasmid
7. Apply to solid medium

The sizes of our DNA fragments are:
Hydramacin-1: 252bp
Sparamosin26-54: 84bp
LL-37: 111bp

Since the DNA contains negative electric charge, we placed the gel with wells toward the negative charge of the buffer tank, as the other side is positively charge, the DNB fragments will run toward that side and separate due to different sizes. The larger the size, the slower the DNA moves. Keep in mind that the TAE buffer needs to fully cover the gel. And also never forget to add marker or you won’t be able to compare the sample with the right band. Run the electrophoresis at constant 150V for 20 minutes.

Before running the electrophoresis, we added DNA loading buffer to the PCR product, according to its concentration.

After the electrophoresis, we put the gel under ultra slim LED illuminator to see the DNA and cut out the gel with DNA fragments for the next step.

We wanted the purified DNA/Gel to insert it into the plasmid---the process is similar to the plasmid extraction.

1. Add 500µL B2 buffer to the gel
2. Water bath at 50℃for 5-10 minutes to make the gel melt in buffer
3. Add around 140µL isopropanol (for the DNA fragments that are less than 500 bp)
4. Move all the liquid into the absorption column with membrane, centrifuge for 30 seconds, 8000Xg
5. Add 500µL Wash Solution, 9000Xg centrifuge for 30 seconds, repeat this step again
6. Add nothing and centrifuge again for 1 minute, 9000Xg
7. Put the absorption column into a clean 1.5mL centrifuge tube, add 15-40µL Elution Buffer, put in room temperature for 1 minute and centrifuge for 1 minute to keep the DNA solution.
8. Test the DNA concentration using Nanodrop machine
    a) Hydramacin-1: 48.6ng/µL
    b) Sparamosin26-54: 29.9ng/µL
    c) LL-37: 28.95ng/µL

○ Positive control --- Kana antibacteria
○ Negative control --- empty pET28a plasmid

○ Hydromasin-1
○ Spheniscin-2
○ LL-37
○ Sparamosin 26-54
○ LL-37-spa

Hydromasin-1 was in puc47 plasmid and we needed to do PCR to take it out and move into pET28a.

We use NcoI and XhoI to cut the pET28a plasmid and connect the DNA fragments in.
We didn’t use T4 connecting enzyme but Gibson since T4 requires restriction sites while Gibson can process homologous recombination.

Materials: 20µL
1. pET28a plasmid 8µL
2. Target gene
3. Gibson 2µL
4. Buffer (5X) 4µL
5. ddH2O
30fmol of pET28a：60fmol of DNA

Plasmid: 30fmol=0.02*n(ng)
5.4kb=5400ng
0.02*5400ng=108ng
108ng | plasmid concentration (ng/µL) --- measured when extracting plasmid = 8µL

Target DNA --- 60fmol=0.04*n(ng)
According to different bp

Kb=kilobase
Bp=base pair

1) Add 0.1µL of DNA
2) According to DNA bp * 0.04/concentration

After adding all the ingredients together(buffer and enzyme last as they are stored in refrigerator)
PCR 37℃ 30min --- let target DNA connect into the plasmid.

1. Precool 1.5ml tube, pET28a plasmid is really sensitive to temperature, easy to die
2. Add 35µL of pET28a into the tube, and 5µL of plasmid, put the 1.5ml tube on the ice. We stored the leftover plasmid at 4℃ in case we need them in the future
3. Put the mix in ice for 30 minutes to fully mix them
4. Water bath at 42℃ for exactly 90 seconds, or else the cell will keep expanding and die, to make competent cell expand, allowing plasmid to enter.
5. Put the tube into ice for 2-3 minutes to shrink the membrane
6. Add 700µL non-kana medium to activate the plasmid, prepare the E.coli for kana selection
7. Put in shaker for 1 hour, 37℃, 220RPM
8. Centrifuge 1000RPM 1 min, precipitate the E.coli, get rid of 650µL of supernatant
9. Resuspend the e-coli and apply to the solid medium

1) Put 50µL of e-coli solution to the solid LB medium petri dish
2) Use apply stick to distribute the solution evenly
3) Raise the e.coli over night in the incubator
4) Get the strain and put into liquid LB with kana
5) Then run colony PCR to see if the DNA is right

1. Take colony and do colony PCR
2. Plasmid Extraction
3. Double Digestion
4. Transforming and applying to the petri dish
5. Run Gel electrophoresis and test sequencing

◇ We added 50µ LB medium with no bacteria and antibiotics into each 1.5mL EP tube
◇ Used tips to take the E.coli colony and put them into EP tubes
◇ Do Colony PCR

1. We added 5µL ddH2O
2. Added 10µL Primer S. Mixer (which includes dNTP, DNA polymerase, and buffer[contains salt to adjust pH])
3. Added 3µL of DNA template
4. Added 1µL of forward and reverse primer

1) 95℃, 5min, pre-degenerate
2) 95℃, 5-10sec, DNA degeneration
3) Mininum Tm value -5℃, 10-15sec, annealing
4) 72℃, time depends on the speed of replication of the enzyme, elongation
5) 72℃, 10min, fully elongate
6) 4℃, infinite time, preserve

After doing the Colony PCR, we wanted to check whether the BL21 was successfully transfected.

Since we used the Gibson enzyme, there are three possibilities for the BL21 on the petri dish to survive (Resistant gene is in the plasmid):
1. No target DNA in BL21, only the plasmid
2. Two plasmids connected with each other
3. Plasmid with target DNA inside, which is what we wanted.

We check the bands after gel electrophoresis to see whether the survived BL21s (another selection) were the plasmid we wanted

We picked three colonies that are supposed to have our target DNA H: A, B, and C
We made TAE solution: 1L in total=980mL ddH2O with 50X TAE buffer

According to the PCR result, A and C had the target DNA we wanted and the according colony can be put into liquid LB to culture

We cultured E.coli with pET28a the previous night and used that to do plasmid extraction The plasmid concentration tested were:

Materials:
For every 50µL system:
1) Plasmid: 1.5µg
2) NcoI: 1µL
3) XHoI: 1µL
4) Cut Smart(50X): 5µL (Buffer)
5) ddH2O: Fill to 50µL
37℃ water bath for 2 minutes

We transformed the Spheniscin-2, Fusion, and DH5a colonies
30µL BL21, target DNA being 1/10 the amount of BL21

Needed to be done in clean bench

10µL system:
1. 1.5µL ddH2O
2. 5µL Primer S. Mixer
3. 2.5µL template DNA
4. 0.5µL each forward and reverse primer

We picked the best ones for sequencing, to verify whether the DNA sequence had the correct DNA

1. Small shake, Test OD₆₀₀, turn to big shake, add IPTG to induce protein expression
2. Protein purification
3. SDS-PAGE Gel Electrophoresis

To amplify the colony by 100 times, and the big shake the next day would amplify it again for 100 times
1. We added 20µL of Kana antibiotics to 20mL of non-antibiotic LB medium
2. Mixed 3.5mL of Kana LB medium (liquid) with 10µL of E.coli solution
3. 220RPM, 37℃ shaker overnight

We poured the melted solid LB medium with Kana antibiotic into the petri dish in the clean bench

We turned the overnight small shake to big shake for around 2 hours

Used OD 600 to check whether the big shake is good for IPTG or not, normally 0.4-0.6 is ideal

Added 350µL IPTG to 350mL of shaked solution with E.coli and LB medium

Mechanism of IPTG:

We needed 20mM of IPTG:
IPTG=238.31g/L=1mol
We needed 10mL of solution thus: 238/31g/L/100=2/3831g --- 1mol
1mol= 50*20mM
So: 2.3831/50=0.047 IPTG

Protein Purification:
Buffer A: Make His tag bind with Ni2
*Low Imidazole 500mL
1. 20mM Na2HPO4 1.42g
2. 500mM NaCl 14.5g
3. 20mM Imidazole 0.68g
4. Adjust the pH to 7.4 by using HCl
5. Add ddH2O to total volume: 500mL

Sample Calculation:
NaHPO4:1L 142g=1mol
500ml 71g=1mol
20mMol=1/50 mol
Thus, 500mL 71/50g=20mMol

Buffer B: Washes off His tag and Ni2 so we can get the antibacterial peptide
* High Imidazole 250mL
1. 20mM Na2HPO4 0.71g
2. 500mM NaCl 7.3g
3. 500mM Imidazole 8.5g
4. Adjust the pH to 7.4 by using HCl
5. Add ddH2O to total volume: 0.25L

We used two 50mL centrifuge tubes to centrifuge the bacteria solution after big shake, repeated the step until all bacteria solution is centrifuges and the E.coli were precipitated to the bottom. (5000 RPM for 10 minutes each time, tube’s precipitate side face up)

We discarded the supernatant, added 5mL of buffer A to tube 1 and vortexed them to resuspend the E.coli, poured the solution to the other tube with E.coli, then added another 5mL of buffer A to tube 1 to fully wash the tube, and added them to the other tube, and resuspended all the E.coli.

We put the tube with buffer A and bacteria in ice and do ultrasonic cell wall breaking. (The ice is to prevent overheating) 40% power, 25℃ temperature, 30 minutes (3 seconds working and 4 seconds resting as a loop)

*Extracted 50µL solution after breaking for future electrophoresis.

We centrifuged the tube for 30 minutes, 12000RPM to separate the cell fragments and solution

Put the tube with supernatant on ice, take 50µL for electrophoresis

Added 100µL of ddH2O to precipitate and vortex to resuspend, take 50µL for electrophoresis

1. Prepare Ni Column --- we used 1mL Ni Beads (Buffer A and B added need to be 10 times of Ni Beads)

Note that never let the solution in the column fully go out or the Ni Column would go dry and unable to be used again.

Column wash: Open both the plugs to let the 20% ethanol solution to drip, then add 10mL ddH2O to drip, and 10mL buffer A to prepare for purification to drip, plug in the plugs to prevent dry out.

1. Add all the supernatant we had into the Ni Column and wait for 10 minutes to let protein and the Ni beads bind. Do not let it drip, prepare a new tube to catch the drip later.

2. Take 50µL of binded solution for electrophoresis

3. Let the solution drip, and then add 10mL of buffer A to wash, collect them in the same tube, and take 50µL of solution from that tube for electrophoresis

4. Plug the bottom plug and add 10mL of buffer B for elution, to elute off the antibacterialpeptide, wait for 3-5 minutes and let it drip. Take a new 15mL tube for collection.

5. Take 50µL for electrophoresis.

6. Store all the solutions on ice, or in the fridge

Remember to rewash the Ni Column for future usage:
1) 5mL buffer A to drip
2) 10mL ddH2O to drip
3) 10mL 20% ethanol and plug both plugs

This Gel is used for running antibacterial peptide samples to see if the thing we purified is the right ones or not

We made 15% lower gel since we had 230 base pairs, aka 76.6 amino acids. Every 20 amino acids have an average of relative molecular weight of 128 (0.128kDa)

Thus, our samples should have about 9.813kDa. And according to the instruction, a 15% lower gel fits the best

Make lower gel:
1. We added 2.7mL of lower gel solution
2. Added 2.7mL of lower gel buffer
3. Added 60µL of coagulant
4. Added the mix to gel making glass plate and add some ddH2O to make the get flat
5. Wait until the gel solidify, pour out the water

Make upper gel
1) Mixed 0.75mL upper gel solution with 0,75mL of colored upper gel buffer
2) Added 15µL of coagulant
3) Plugged in the well comb
4) Waited until the gel solidify and plug out the comb, used paper towel to wrap around the glass plate and added ddH2O to keep the gel moistured

We vertically assembled the gel cassette with the chamber and the buffer tank, added buffer into the chamber to check whether it leaks or not, then added recycle buffer into the tank to let electricity able to pass through.

For the wells on the gel, we added the samples in the sequence as follows for all 5 genes and empty pET28a:

1. Marker
2. Crude extract (Solution after ultrasonic cell wall breaking)
3. Supernatant (Top liquid after centrifuge)
4. Precipitation (Precipitate after centrifuge and re-suspended)
5. Mixture solution (After supernatant combine with nickel column)
6. Washing liquid (After washed by buffer A)
7. Eluent (After washed by buffer B)
Run 60V for 30min and then 120V for 1 hour

For sample 2-6, we mixed 1µL of sample with 7µL buffer B and 2µL 5X loading buffer since the concentration of the samples were high and needed to be diluted by buffer B. For sample 7, we simply mixed 8µL of solution with 2µL loading buffer.

Then we put the 2-7 PCR tube into PCR machine, 95℃ for 5 minutes to denature the protein and able to run the SDS-PAGE Gel Electrophoresis

1. We poured the running buffer back to the recycle bottle
2. Took out the get, cut off and discarded the top gel with wells, used coomassie brilliant blue stain to cover the gel
3. Put the gel on the shaker overnight (if time is limited, head to 60℃ and shake for 2 hours) to color the protein with stain, anything that is not protein would not show color, or at least we cannot see
4. Used detergent to wash off the stain
a) Detergent: 100µL of methanol, 100µL acetic acid, and 800 µL ddH2O

Original protein solution
We dilute from original to (time of dilution): 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024
300µL of LB medium in each tube to dilute

We had three bottles of water with bacteria from different fish tanks in total, so we mixed them together to 2mL and added 18mL of LB solution to dilute them for 10 times

Except for the compare groups, each well we added 100µL of bacteria solution and 100µL of protein we got

For the four compare groups, the systems are as follows:

· Blank control: 200µL of LB medium
· Environmental Control: 100µL of LB medium and 100µL of bacteria solution
· Negative control: 100µL of LB medium and 100µL of empty pET28a protein
· Positive control: 100µL of bacteria solution and 100µL of 10X kana antibiotic

Shake at 37℃, 220 RPM for 12 hours and then use microplate reader to test absorbance. The less the number, the less the bacteria growth.