Project : DNA Cloning
Saturday, 2019-7-20 ———————————————-
PCR amplification of MlrA 1, Mlr2, and MlrA3.
This experiment is to verify if the DNA sequence is suitable for protein expression.
The process of creating the reaction system is as the following.
- Add 18 ul of water
- 25 ul of 2 x Phanta Buffer
- 1 ul of dNTP
- 2 ul for each of the two primers
- 1 ul of the template
- 1 ul of the DNA Polymerse
Next, put reaction tube through the PCR machine.
Add 0.5g Agarose with 50ml 1xTAE Buffer to get 1% gel. Microwave to melt the solution and add 1microliter of nucleic acid pigment. Then, add the matrix and wait till the gel concrete.
Recycle the PCR product
A. Add 5 microliter of 10 x loading buffer to the PCR system, perform agarose gel electrophoresis to factional DNA fragments. Conduct electrophoresis with 110 volts of electricity for 30 minutes, Image formation of gel on computer.
B. Sever the target sectors from the gel
C. Add 350 microliter of binding buffer, heat the product at 65 ℃ till the gel melts.
D. Transfer the solution into the binding column.
E. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 1 minute.
F. Discard the yellowish waste solution.
G. Add 500 microliter of wash buffer.
H. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 1 minute.
I. Discard the yellowish waste solution.
J. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 2 minutes.
K. Dry the binding column under room temperature.
L. Add 30 microliter of 65 degrees celsius heated deionized water.
M. Cap the solution, keeping the solution at room temperature, right until the solutes are uniformly dissolved.
N. Revolve the solution in the centrifuge under 12000 rpm of revolution rate for 2 minutes, separating the DNA.
Use nanodrop to measure the concentration of DNA.
This is the formation of gel seen under an ultraviolet light.
The final concentration is 29.2 ng / 10^-6L
Sunday, 2019-7-21 ————————————————
LB Culture Medium
A. Prepare 100ml solid culture medium & 600ml liquid culture medium
Solid Medium - Tryptone 1 g + Yeast Extract 0.5 g + Sodium Chloride 1g
Liquid Medium - Tryptone 6 g + Yeast Extract 3 g + Sodium Chloride 6 g + Agar 6-7.5 g.
Use restriction enzymes to cut the plasmid.
Linearizing the Plasmid using restriction enzymes.
A.1% gel: 0.25g Agarose + 25ml 1xTAE Buffer + heat, melt the solution, add 1microliter of nucleic acid pigment. Then, add the matrix and wait till the gel concretes.
Linearized pTOLO - Ex5
B.Conduct electrophoresis with 110 volts of electricity for 30 minutes.
C.Sever the target sectors from the gel.
D.Collect the plasmids.
Homologous RecombinationPrepare LB Plate
Melt the solid medium using the microwave.
A. Add 50 ml 2000 x Kanamycin Monosulfate into 100 ml melted solid medium
B.Pour the mixture into separate plates and wait for it to concrete.
A.10 microliter homologous recombination product + 50 microliter competent cell, mix the solution
B. Set the solution on ice and wait for 30 min
C. 42 degrees celsius Heatshock for 45 sec
D.Place it on ice for 2 min.
E.Add 300 microliter LB liquid culture medium, place them inside the incubator at 37 degrees celsius at 220 RPM for an hour.
F.Spread the medium evenly on the surface plate.
G.Invert the plates and incubate at 37 degrees celsius overnight.
Monday, 2019-7-22 ————————————————
Find monoclonals on the petri dish and move them into small test tubes.
a.Add 2.5 ul 2000x of Kanamycin Monosulfate to the 5 ml liquid LB medium.
b.Use a sterilized toothpick to find the monoclonal.
c. Shake for at least 10 hours at 220 rpm (37°)
This is an image of the Petri dish.
Use PCR to determine the positivity of the plasmid.
Use 2x mix-taq enzymeProgram ：
2x mix - tag 10 ul
T7-F primer 2ul
MlrA 2/3 2ul
Template（2ul Bacterial Fluid）
DD water 4 ul
95° for 3 minutes
72° for 80 seconds
72° for 5 minutes
4° for ∞
Draw the plasmid
a.Pour the bacterial fluid into a 2ml EP tube.
b.Centrifuge for one minute at 12000 rpm.
c.Discard the supernatant.
d.Add 250 ul of solution 1.
e.Vortex oscillation to make the cells suspend.
f.Add 250 ul of solution 2.
g.Mix the EP tube upside down.
h.Add 250 ul of solution 3.
i.Mix upside down several times to form precipitate.
j.Centrifuge for ten minutes at 12000 rpm.
k.Aspirate supernatant, add Binding Column.
l.Centrifuge for one minute at 1200 rpm.
m.Discard the liquid.
n.Add 500 ul of wash buffer.
o.Centrifuge for one minute at 12000 rpm.
p.Discard the liquid
q.Centrifuge for two minutes at 12000 rpm, dry at room temperature for two minutes.
r.Add 30 ul of DD water, set aside for two minutes to fully dissolve the plasmid.
s.Centrifuge for two minutes at 12000rpm, elute the plasmid.
t.Use Nanodrop to test the plasmid concentration.
u.Take 5 ul of plasmid to measure the concentration at the company.(Plasmid concentration higher than 100 ug/ul）
v.Transform the correct plasmid to BL21 DE3 competant cells（0.5ul）
Produce medium （9 Liters of LB medium）
Prepare LB Plate
Produce medium （9 Liters of LB medium）
Fetch bacteria sample from a single bacteria colony.
A.Till the bacteria finished incubating, place them inside the large flask.
B.Inject 500microliter of 2000x Kanamycin Monosulfate into the large flask with 1L of capacity
C.Place the solution in the centrifuge at 220 rpm 37 degrees Celsius till OD reaches the range of 0.6~0.8
D. Lower the temperature to 18 degrees Celsius, add 350microliter of 1 mole IPTG
Place the solution in the centrifuge at 190 rpm 18 degrees Celsius to make the bacteria to express the protein for 14 to 16 hours.
(2) Protein Expression Testing
A.Inject 3ml of lysis buffer into the cells
B.Conduct Ultrasound Bacteriolysis at 20percent rate of work for 10 min
C.After lysis, take 20microliter as template and another 50microliter into centrifuge at 12000rpm for 2 min.
D.Draw the supernatant liquid out after using the centrifuge, consider it as one of the sample
E.Place 20 microliter of buffer and resuspend, consider it as one of the sample
F.Fetch the sample before adding IPTG
G.Add 5 microliter loading buffer into all the samples.
I.Dye and decolorize the gel, and assess the situation
This was the SDS - Page Gel that we dyed and decolorized.
Collect BL21 DE3 cells
Fetch bacteria sample from a single bacteria colony.
A.Add 15 mL bufferA, which contains 20 mM tris-chl and 100 mM NaCl, into 1 L E.coli cells pretreated by centrifugation. Then resuspend the cells
B.Add DNA polymerase, MgCl2, PMSF containing 10 mg/ml of 2000x mother liquor of DNA polymerase, 2 M of 2000x mother liquor of MgCl2, 1M of 1000x mother liquor of PMSF.
C.French press with pressure reaching 15,000 psi and lyse the cells twice.
D.Centrifuge the cells with 15,000g centrifugal force for 10 minutes.
E.Collect the supernatant of the cells, and then ultra-centrifuge the supernatant with 150,000g centrifugal force for an hour.
F.Discard the supernatant after ultra-centrifugation. Add 6 mL buffer A per liter of cells into the sediment, and then resuspend. Then add DDM, a detergent, to a final concentration of 1 %. Incubate the solution under 4 degrees Celsius for 2 hours.
G.Centrifuge the solution with 20,000g centrifugal force for 45 minutes.
H.Collect the supernatant after centrifugation and bind the supernatant with Ni21-NTA. Add 1ml resin per liter of cells. Then incubate overnight.
Further purification of MlrA protein using nickel column
A.The protein that binds to Ni21-NTA is added to the affinity column. The liquid that flow through is collected.Using the method of gradient elution, while the amount of imidazole gradually increases, the amount of DDM simultaneously decreases.
Ultimately, the final amount of imidazole reaches 200mM，and the percent of DDM within the affinity column reaches 0.018%
URun SDS-PAGE Gel to identify the results of protein purification.
The followings are the samples for Gel electrophoresis：
1. The total sample of pTOLO-EX5-mlrA 2/3 in BL21(DE3) after the lysis of BL21 (DE3) cells
2. The sample of precipitants after cell lysis and low-speed centrifugation
3. The sample of supernatant after cell lysis and low-speed centrifugation
4. The sample of supernatant after ultra-centrifugation
5. The total sample after the membrane lysed
6. The sample of precipitants after the membrane lysed
7. The sample of supernatant after the membrane lysed
8. The sample of impure proteins that first flow through the nickel column
9. The sample after 5mM imidazole wash
10. The sample after 10mM imidazole wash
11. The sample after 20mM imidazole wash
12. The sample after 200mM imidazole elution
According to the results of Gel Electrophoresis, there is very few amount of proteins shown at the target strip. While on the contrary, we observed high concentrated protein in the impure proteins that first flowed through the nickel column and in the supernatant after the membrane lysed, though the size of the protein was slightly smaller than our target protein. We hypothesized that the protein with higher concentration was probably the MlrA without the sumo tag, otherwise it would just be another impure protein. Because of this uncertainty, we decided to react the impure proteins that first flowed through the nickel column and the supernatant after the membrane lysed with cyanotoxin separately. The results of the reaction will verify whether the two samples contains MlrA or not.
Figure. 1 pTolo-EX5-MlrA3-detergent-Ni
Figure. 2 pTolo-EX5-MlrA2-detergent-Ni
Figure. 4 pTolo-EX5-MlrA2-detergent
24-hour reaction of MlrA with cynaotoxin
aBesides using the supernatant after the membrane lysed and the impure proteins that first flowed through the nickel column to react with cyanotoxin, we also tested the purified MlrA 3 with 2mg/mL as its initial protein concentration. We tested 13 different reaction in total. As independent variables, the concentration of the target protein and the concentration of cyanotoxin were adjusted differently for each of the 13 reactions. The temperature was fixed at 27 degrees Celsius and the reaction time was fixed at 24 hours.