Team:SUSTC-Shenzhen-B/protocol1

Lab Protocol

Brief Process

1. Site-Directed Mutagenesis

2. Restriction enzyme digestion and electrophoresis

3. Media Preparation

4. Bacterial Transformation

5. Colony PCR for verification

6. Cultivate the bacteria

7. Plasmid DNA Isolation

8. Restriction Enzyme Digestion and Electrophoresis

9. Polymerase Chain Reaction and electrophoresis

10. Double Restriction Enzyme Digestion and Electrophoresis

11. Ligation

12. Bacterial Transformation

13. bacterial colony PCR

14. Cultivate the bacteria

15. Plasmid DNA Isolation

16. Restriction Enzyme Digestion and Electrophoresis

17. Ligation

18. Bacteria transformation

19. Cultivate the bacteria

20. Flow Cytometer Analysis

21. Fluorescence Microscope

Site-Directed Mutagenesis

Plasmid psb1a3 is chosen to be the vector that ligates GPF and RFP fragments.To protect the structural integrity of the constructed plasmid, a restriction enzyme cutting site named Pst I need to be mutated to Afl II. Proper primers are designed for this purpose.

Method

1.

• Prepare the sample reaction as indicated below:

Total: 25μl
+  0.25 μl of Ex Taq polymerase
+  2.5 μl of 10× Taq reaction buffer
+  2.0 μl  of dNTP(2mM)
+  1.0 μl of template (E.coli plasmid 817)
+  1.0 μl of oligonucleotide primer PtoA-F
+  1.0 μl of oligonucleotide primer PtoA-R
+  18.25 μl of ddH2O
Note: Here listed the primers and their sequences.
PtoA-F  5'-CCACCTGACGTCTAAGAAAC-3'
PtoA-R  5'-ATGATCATCGCCGGCGAATTCAGGC-3' 
2.  Set thermocycler temperatures and the time.
Procedures on the thermocycler are listed below:
① 94˚C for 5 min
② 30 cycle
a. 94˚C for 1 min
b. 55˚C for 1 min
c. 72˚C for 1 min20sec
③ 4℃ for 7 hours

Restriction Enzyme Digestion for Verification

In the condition of restriction enzyme cutting site is mutated correctly, a special step to proof the result is in need. A restriction enzyme digestion can be executed and result can be revealed by electrophoretogram. We used restriction enzyme Afl II digestion as a sample group and Spe I digestion as a control group.
Method
1.  Prepare the control reaction as indicated below:
Total: 10μl
+ 0.5μl of Pst I restriction enzyme (company :Takara)
+ 1μl of 10XH buffer
+ 1μl of plasmid DNA
+ 7.5μl of ddH2O
2.  Prepare the sample reaction as indicated below:
Total: 10μl
+ 0.5μl of Afl II restriction enzyme, (company :Takara)
+ 1μl of 10XM buffer
+ 1μl of plasmid DNA
+ 1.0μl of 0.01% BSA
+ 6.5μl of ddH2O
3. Put the tubes in 37℃ water bath for 1-2h.
4. Prepare electrophoresis gel by adding 0.6g agarose to 60ml TAE (1% solution,1X,diluted from 50X TAE). Pour on conical flask and cover the Conical flask sealing surface with silver paper to avoid the loss of water vapor. Place in the microwave and microwave on middle for 1 minute at a time, pulling it out and swirling until solution is homogeneous again, then repeat(BE CAREFUL to watch the solution closely when swirling–it superheats and can boil over and cause severe burns). Continue until solution is seen clear and homogeneous with no existence of solid.Add 3 μl of Gelred ( 10000X ) .  
5.  By inserting the pipette tip below the TAE liquid and into the well, add 5μl of 1kb DNA ladder solution to first (and last if desired) well, skip one well, then begin adding the 5μl of digested DNA solutions mixed with 1μl loading buffer (6X) to the wells.
6.  Place the cover on the electrophoresis unit, plug into the power source, and turn on voltage to 120V, set time to 30 minutes, and press the start button twice,until the bubbles are seen. DNA separation can be observed as time goes on by turning off the power supply then gently removing the basin from the electrophoresis unit (be careful not to let the gel slip out of the basin) and placing on the UV transilluminator to see DNA bands.
7.  When the desired level of separation is obtained, the basin can be placed on the transilluminator for picture taking(Of the absence of transilluminator,we use camera to take pictures with the UV light ).
8.  Cut the gel of specific position and collect it in tubes that have measured weight.
9.  Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3.
Figure

(figure 1 : This figure shows that the site-directed mutagenesis succeed ,we successfully change a restriction enzyme cutting site named Pst I to Afl II. Lane 1 represents the plasmid mutant-psb1a3, lane 2 shows that the mutant-psb1a3 cannot be digested by restriction enzyme Spe I ,lane 3 shows that mutant-psb1a3 can be digested by restriction enzyme Afl II.)

Media Preparation

For all experiments involving the bacterial biomass and experimentation, proper media is chosen to grow the cells. Commonly,we use Lysogeny broth media for E. coli. The following is the media compositions and their quantities.
Lysogeny Broth (LB) liquid media (1 L)
Measure out these following:                                                                     

1. Bacto-Tryptone - 10 g
2. NaCl - 10 g
3. Yeast Extract - 5 g

Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.
Lysogeny Broth (LB) solid media (1 L)
Measure out these following:

1. Bacto-Tryptone - 10 g
2. NaCl - 10 g
3. Yeast Extract - 5 g
4. Difco Agar - 15g

Add ddH2O and 5mmol/L Tris Buffer in a measuring cylinder to ensure accuracy, to make a total of 1 liter and pH is 8.0.
Autoclaving

• Autoclave at 121 °C for 60 minutes. After the media cooling down enough, antibiotics Ampicillin(100mg of Ampicillin per 1ml of the media) are added. At last the media are poured 15ml on each plate and become solid.Store the plate at 4℃ refrigerator.

 

Bacterial Transformation

Introduction of exogenous DNA into cells using non-viral methods is called “Transformation”.Transformation is commonly used to introduce recombinant plasmid DNA into bacterial strains which can transform naturally or can be made competitive for transformation by artificial means.
Depending on the expected transformation efficiency, there are two main types of competent cells that can be used for transformation.
1.Chemically competent cells
Chemically induced competent cells are calcium chloride-treated to facilitate attachment of the plasmid DNA to the competent cell membrane. During chemical transformation, the cells are heat-shocked in a water bath; which opens the pores of the cell membrane allowing entry of plasmid DNA from the buffer.
2.Electrocompetent cells
Electrocompetent cells are prepared for transformation using electroporation, a method that uses an electrical pulse to create pores through which genetic material enters the cells. This method usually has high transformation efficiency.
Method
1. Take out an appropriate number of tubes that contain competent cells(100μl ) from the freezer. Immediately place the tubes on ice, so that all but the cap is surrounded by ice. Allow the cells to thaw on ice for 2-5 min.
2.  Visually check the cells to see whether they have thawed and gently flick the cells 1-2 times to evenly resuspend the cells.
3. Add 10μl PCR products(mini-prep purified) to the competent cells DH-5α. Stir gently to mix and return the tube to the ice, making sure that the tube is surrounded by ice except for the cap. Repeat for additional two times for the same samples.
4. Incubate the tubes on ice for 30 min.
5. Place the tubes in a 42°C water bath for exactly 90 sec; do not shake.
6. Place the tubes on ice for 2 min to cool down.
7. Add 800 l of room temperature LB medium to each tube.
8. Shake the tubes vigorously at 37°C for 45-60 min.
9. Centrifuge the tubes at 3K RPM for 1 min. Discard the supernatant liquor and leave 100-200 μl of the mixtures.Mix the contents and spread the whole liquid on LB agar plates containing the appropriate antibiotic ampicillin for the plasmid.
10. Place the plates on the bench for several min to allow excess liquid to be absorbed, and then invert and incubate overnight at 37°C (12-16 h).

Colony PCR for verification

Colony PCR is used to identify and select cell colonies that have the correct plasmid inserted. The procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up several colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.
Method
1. Prepare the sample reaction as indicated below:
Total: 25μl
     +  0.25 μl of Ex Taq polymerase (company:Takara)
     +  2.5 μl of 10× Taq reaction buffer
     +  1.0 μl of R-NPS-F
     +  1.0 μl of G-SXA-R
     +  1.0 μl of plasmid mutant-psb1a3
     +  2.0 μl of dNTP( 25mM )
     +  17.25 μl of ddH2O
2.  Procedures on the thermocycler are listed below:
   ① 94˚C for 5 min
      ② 30 cycle
       a. 94˚C for 1 min
       b. 55˚C for 1 min
         c. 72˚C for 1 min20sec
      ③ 4℃ for 7 hours
3.  Electrophorese the total system and observe the lane separation.

Cultivate the Bacteria

According to the results of the   PCR detection, positive colonies are chosen and transferred them to 5ml LB liquid media ( 5μl of ampicillin added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.

Plasmid DNA Isolation

Use E.Z.N.A.TM Plasmid Mini I to realize plasmid DNA isolation.
Method

• Transfer 5 ml of overnight culture into a 1.5-ml eppendorf tube labeled with group number. 
• Centrifuge the sample at max. speed of desk top centrifuge and RT for 1min to pellet the cells.
• Discard the supernatant. Remove as much of the supernatant as possible without disturbing the cell pellet.
• Repeat step 1 and 2 twice.
• Resuspend the pellet completely in 250 ml of Solution I (containing RNase A) by vortexing the samples vigorously . No clumps should be visible in the tube.
• Add 250 ml of Solution II and mix the sample by gently inverting the tube 4 to 6 times. Do not vortex or shake the sample vigorously. The bacterial suspension should begin to clear which have lysed the bacterial cells in this step. Warning: Do not stop here for more than five min, as the high pH hurts your DNA!
• Add 350 ml of Solution III and mix by gently inverting the tube 4 to 6 times until a flocculent white precipitate forms. Do not shake vigorously, as it might break the genomic DNA.
• Centrifuge at maximum speed for 10 min at room temperature to pellet the cell debris. You should see a white precipitate in the tube after the centrifugation.
• While the samples are centrifuging, for each sample, label a clean HiBind Miniprep Column which is to assembled in a 2-ml collection tube
• Apply the supernatants from step 8 to the columns.
• Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube.
• Add 500 ml of Buffer HB to wash the Hibind Miniprep Column. Centrifuge at maximum speed for 1 min at RT. Discard the flow-through in the collection tube. 
• Wash the column by adding 700 ml of DNA Wash Buffer diluted with absolute ethanol. Centrifuge at maximum speed for 1 min at room temperature and discard the flow-through.
• Then centrifuge the tubes again for 2 min to remove all the moisture.
• Place the column in a clean 1.5 ml eppendorf tube that is labeled with the plasmid name and group number. To elute the DNA, add 50 ml of Elution Buffer to the center of each column. Let the samples stand for 2 or more minutes at RT, and then centrifuge for 1 min. The sample in the centrifuge tube (bottom) is your plasmid DNA.
• Discard the column and save the sample in the eppendorf tube by placing it in the freezer (-20°C). 

Restriction Enzyme Digestion and Electrophoresis

Because the colony PCR test is so sensitive and affect markedly by environment factors. So we do a restriction enzyme digestion to ensure that the isolated plasmid is the site-directed mutated plasmid.
Method
1.  Prepare the control reaction as indicated below:
Total: 10μl
     + 0.5μl of Pst I restriction enzyme(company :Takara)
     + 1μl of 10XH buffer
     + 1μl of plasmid DNA
     + 7.5μl of ddH2O
2.  Prepare the sample reaction as indicated below:
Total: 10μl
    + 0.5μl of Afl II restriction enzyme, (company :Takara)
    + 1μl of 10XM buffer
    + 1μl of plasmid DNA
    + 1.0μl of 0.01% BSA
    + 6.5μl of ddH2O
3.  Electrophorese the total system and observe the lane separation.

Polymerase Chain Reaction(GFP & RFP) and electrophoresis

GFP and RFP DNA fragments are the insert which need to be ligate to the plasmid mutant-psb1a3. Do a PCR amplification can get enough quantities for the following reactions.
Method
１．Prepare the sample reaction as indicated below:
Total: 100μl ( PCR amplification of GFP fragments）
   + 1.0μl of Taq DNA polymerase,#EP0402
   + 10μl of 10XTaq buffer
   + 10μl of MgCl2(25mM)
   + 10μl of dNTP(2mM)
   + 2μl of G-SXA-R
   + 2μl of G-SXA-F
   + 4μl of DNA template
   + 61μl of ddH2O
Total: 100μl（PCR amplification of RFP fragments）
   + 1.0μl of Taq DNA polymerase, EP0402
   + 10μl of 10XTaq buffer
   + 10μl of MgCl2(25mM)
   + 10μl of dNTP(2mM)
   + 2μl of R-NPS-R
   + 2μl of R-NPS-F
   + 4μl of DNA template
   + 61μl of ddH2O
Note:  Here listed the sequences of primers.
R-NPS-F   5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' 
R-NPS-R   5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3'
G-SXA-F   5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' 
G-SXA-R   5'-CCGACTTAAGGGATCCTATAAACGCAG-3'
２．Procedures on the thermocycler are listed below:
     ① 94˚C for 5 min
      ② 30 cycle
       a. 94˚C for 1 min
       b. 55˚C for 1 min
         c. 72˚C for 1 min20sec
      ③ 4℃ for 7 hours

• Use  DNA Gel Extraction Kit to purify the GFP and RFP DNA fragments after the Electrophoresis.

Figure

(figure 2 : This figure shows that The PCR reaction system can amplify large quantities of GFP and RFP DNA fragments. The digestion based on the GFP and RFP DNA fragments can be done to prepare for the ligation. Lane 1 represents the template E.coli 817 can amplify the GFP and RFP DNA fragments , lane 2 represents the template E.coli 817(355.5) can also amplify the GFP and RFP DNA fragments.)

Double Restriction Enzyme Digestion and Electrophoresis.

Use specific restriction enzymes to digest plasmid mutant-psb1a3,GFP and RFP to get sticky ends and purify the DNA fragment after the Electrophoresis.
Method:

•  Digestion of plasmid mutant-psb1a3

Prepare the sample reaction as indicated below:
Total: 50μl
+ 3.0μl of Not I restriction enzyme, #ER0591
+ 3.0μl of Afl II restriction enzyme, #ER0831
+ 5.0μl of 10X buffer O
+ 1.0μl of mutant-psb1a3 plasmid
+ 37.0μl of ddH2O
2.  Digestion of PCR product GFP
Prepare the sample reaction as indicated below:
Total: 50μl
+ 3.0μl of Not I restriction enzyme, #ER0591
+ 3.0μl of Spe I restriction enzyme, #ER1251
+ 5μl of 10X buffer Tango
+ 10μl of PCR products GFP
+ 29μl of ddH2O
3.  Digestion of PCR product RFP
Prepare the sample reaction as indicated below:
Total: 50μl
+ 3.0μl of Afl II restriction enzyme, #ER0831
+ 3.0μl of Spe I restriction enzyme, #ER1251
+ 5μl of 10X buffer Tango
+ 10μl of PCR products RFP
+ 29μl of ddH2O
4.   Put the tubes in 37℃ environment for 4-8 hours
5.  Use DNA Gel Extraction Kit to purify the mutant-psb1a3 fragment, GFP and RFP after digestion and named them by mutant-psb1a3 (NA) ,GFP(NS) and RFP(AS) after the Electrophoresis.

Ligation

Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3,GFP and RFP DNA fragments,also ligation can be done. We ligate mutant-psb1a3 vector and sticky GFP and RFP DNA fragments to construct an new plasmid mutant-psb1a3-GR. 
1.  Prepare the control reaction as indicated below:
Total: 10μl
+ 1.0μl of plasmid mutant-psb1a3 (NA)
+ 3.0μl of GFP(NS)
+ 3.0μl of RFP(AS)
+ 2.0μl of T4 DNA Ligase,#EL0011
+ 1.0μl of 10XT4 Ligase buffer
Note: GFP(NS) means the product of GFP DNA fragments digested by restriction enzyme Not I and Spe I.
2.  Prepare the sample reaction as indicated below:
Total: 10μl
+ 1.0μl of plasmid mutant-psb1a3 (NA)
+ 2.0μl of T4 DNA Ligase, #EL0011   
+ 1.0μl of 10XT4 Ligase buffer
+ 6.0μl of ddH2O
3.  Put the tubes in 22℃ water bath, react for 8-12 hours.

Bacterial Transformation

Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours.

Bacterial Colony PCR

Colony PCR is used to identify and select cell colonies that have the correct plasmid insert. This procedure is a way to do several PCR operations on cell colonies in parallel, to evaluate the results and select the corresponding good cell colonies. After an overnight growth of E.coli, we can pick up some colonies from the plate and do a colony PCR verification. Besides, the colonies we choose and should also be stored, we can incubate these colonies in one plate after every colony has been marked.
Method
1. Prepare the sample reaction as indicated below:
Total: 20μl
    +  0.25 μl of Ex Taq polymerase,#EP0402
    +  2.0 μl of 10× Taq reaction buffer
    +  1.0 μl of R-NPS-F
    +  1.0 μl of G-SXA-R
    +  5.0 μl of bacterial colony
    +  2.0 μl of dNTP( 25mM )
    +  8.75 μl of ddH2O
Note: Here listed the sequence of primers.
 R-NPS-F   5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3' 
R-NPS-R    5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3'
G-SXA-F    5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3' 
G-SXA-R    5'-CCGACTTAAGGGATCCTATAAACGCAG-3'
2.  Procedures on the thermocycler are listed below:
     ① 94˚C for 5 min
      ② 30 cycle
       a. 94˚C for 1 min
       b. 55˚C for 1 min
         c. 72˚C for 1 min20sec
      ③ 4℃ for 7 hours

•  Electrophorese the total system and observe the lane separation.

Figure

(figure 3;the lane on the figure are 2k fragments, it shows the GFP and RFP are ligated to the vectors which was isolated from the bacterial colonies.)

Cultivate the Bacteria

According to the results of the   PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.

Plasmid DNA Isolation

Use E.Z.N.A.TM Plasmid Mini I to isolate the constructed plasmid mutant-psb1a3-GR.

Restriction Enzyme Digestion and Electrophoresis

From the last step, we got the certain quantities of isolated plasmids. In this step, we do two restriction enzyme digestion reactions, one to prove that the plasmid is construct correctly ( mutant-psb1a3-GR ), one to get sticky ends preparing for the ligation.
Method

• Restriction Enzyme Digestion to prove that plasmid is constructed correctly

a.  Prepare the sample reaction as indicated below:
Total: 10μl
+ 1.0μl of Not I restriction enzyme,#ER0591
+ 1.0μl of Spe I restriction enzyme,#ER1251    
+ 2.0μl of Buffer Tango( 10X )
+ 1.5μl of plasmid mutant-psb1a3-GR
+ 14.5μl of ddH2O
b.  Electrophorese the total system and observe the lane separation.

• Restriction Enzyme Digestion to get sticky ends preparing for the ligation.

a.  Prepare the sample reaction as indicated below:
Total: 50μl
+ 5.0μl of Pst I restriction enzyme, #ER0611
+ 5.0μl of Xba I restriction enzyme, #ER0681   
+ 3.0μl of Buffer Tango( 10X )
+ 5.0μl of plasmid mutant-psb1a3-GR
+ 32.0μl of ddH2O
b.  Electrophorese the total system and observe the lane separation.
c.  Cut the gel of specific position and collect it in tubes that have measured weight.
d. Use DNA Gel Extraction Ki to purify the plasmid DNA mutant-psb1a3-GR(PX) .
Note: Mutant-psb1a3-GR(PX) means plasmid Mutant-psb1a3-GR digested by Pst I and Xba I.

Figure

(Figure 4 : The double digestion of mutant-psb1a3 forms a linear DNA fragments and it runs slower than circle DNA fragments. This suggest that the double digestion of mutant-psb1a3 works in a high efficiency, and desired sticky ends are formed.1,3,5  are plasmids digested by restriction enzyme Pst I and Xba I from different colonies, 2,5,6 are pure plasmid mutant-psb1a3-GR, 7 is the plasmid mutant-psb1a3. )

 

Ligation

Ligation is the process that target DNA gene is inserted into a plasmid. Both the vector and insert are prepared to have the sticky ends. These two kinds of DNA pieces are placed in a reaction tube and the proper DNA ligase, buffer, and cofactors are added for the reaction to take place. When done properly, the ligation will result in a successful combination of the insert and plasmid into one plasmid.Based on the digestion of mutant-psb1a3-GR, terminator DNA fragments,ligation can be done. We ligate mutant-psb1a3-GR vector and sticky terminator DNA fragments to construct an new plasmid mutant-psb1a3-GR-t.By detecting the quantities of GFP and RFP, terminator efficiency can be calculated.
1.  Prepare the control reaction as indicated below:
Total: 10μl
     + 1.0μl of plasmid mutant-psb1a3 (NA)
     + 6.0μl of terminator
     + 2.0μl of T4 DNA Ligase , #EL0011
     + 1.0μl of 10XT4 Ligase buffer
2.  Put the tubes in 22℃ water bath, react for 8-12 hours.

Bacteria transformation

Transform the ligation products into the DH-5α competent cells, put the plate on 37℃ gas bath for 12-16 hours.

Cultivate the Bacteria

According to the results of the   PCR detection, we choose positive colonies and transfer them to 5ml LB liquid media ( 5μl of ampicillin has added) stored in 12.5ml centrifuge tubes. Put the centrifuge tubes in 37℃ gas bath overnight.

Flow Cytometer Analysis

  1. NaCl solution( 0.9% )
2. 75% Methanol
B.  Procedures:
1. Transfer overnight suspension culture to 1.5ml centrifuge tubes and centrifuge at 12000RPM for 30s. Pour the supernatant and add overnight suspension culture and centrifuge again until enough bacteria has been collected.
2.  Use NaCl solution(0.9%) to mix the bacteria and vibrate the centrifuge tubes until the bacteria distributed uniformly.
3.  Put the centrifuge tubes into the Flow Cytometer and set parameters and run the program.

 

Fluorescence Microscope

Method
Add 10μl of former bacteria solution to micro slide and cover with coverslip.Then placed it on the Fluorescence Microscope with 488nm light activating and observe the GFP and RFP.