7.1.1. Plasmid extraction

Reagents: LB culture medium, Buffer S, Buffer SP1(50 mM glucose / 25 mM Tris-HCl/ 10 mM EDTA,pH 8.0, RNase A), Buffer SP2(0.2 N NaOH / 1%SDS), Buffer SP3(Potassium Acetate/2M acetic acid/75% alcohol)
Purpose: To purify and isolate pUC57 plasmids from E. coli colonies
  1. Extract 1ml of all four E. coli inoculum (excluding cas12a) from the test tubes on the clean bench; preserve them in separate tubes for future experimentation. To the remaining test tubes of each E. coli solution (4ml left in each):
  2. Separate solutions into two 2ml microtubes; centrifuge the microtubes at 8000×g for 2 minutes.
  3. Discard the supernatant.
  4. Add 50 μl buffer S* to each spin column.
  5. Centrifuge the spin column at 12000 ×g for 1 minute.
  6. Add 250 μl buffer SP1 to the microtubes; resuspend the pelleted bacterial cells using a pipette.
  7. Add 250 μl buffer SP2 to the microtubes; mix the tubes backward very slowly and gently 6 times.
  8. Add 350 microliters of buffer SP3; mix the solutions in the same way as in step 7.
  9. Centrifuge the microtubes at 12000 ×g for 10 minutes.
  10. Transfer the mixture into the spin column using a pipette; centrifuge at 8000×g for 1 minute; remove the flow-through.
  11. Add 500ml of Buffer DW1 to the spin column; centrifuge at 9000×g for 30 seconds; remove the flow-through. (Note: this washes off unwanted proteins and DNA adsorbed by the column.)
  12. Add 700μl wash solution (contains absolute ethanol); remove the flow-through. (Note: This step removes the insoluble impurities.)
  13. Repeat the previous step.
  14. Centrifuge the empty spin column at 9000 rpm for 1 minute. (Note: this removes the left-over alcohol on the surfaces.)
  15. Add 30μL of elution buffer (it can also be substituted with ionized water or ddH2O ) onto the membrane of the column; centrifuge at 9000 rpm for 1 minute.
  16. The four types of plasmids are collected in 4 spin columns.

*Buffer S was added to enhance the DNA’s adherence to the spin column’s wall
**Buffer SP1 was added because RNase A triggers RNA degradation
***Resuspending help increase the surface area of contact between the pellets and the reagents
****Buffer SP2 help make the mixture homogenous without breaking target DNA strands (plasmids). As linear bacterial chromosomes (compared to circular plasmids) are more easily dissociated and adsorbed by proteins, they will also form precipitates but circular plasmids will not.
*****Buffer SP3 is a buffer with strong acidity that will separate the testing solution into two distinct sections: a turbid mix of complex proteins on the top and a clear solution of DNA on the bottom. Furthermore, its acidity neutralizes the solution and prevents alkali from causing DNA degradation.

7.2.1. Measure the concentration of four purified pUC57 plasmids (cagA/ipaH/invA/16S integrated plasmids)

Same procedure as “B. Test OD value of pET-28a integrated E. coli (dedicated to produce Cas12a protein)”, except set ddH2O as the controlled group.

Results of the test are shown in the table below:

Integrated Bacterial Sequence Data collected
ng/μl * A260/A280** A260/A230*** A260****
invA 270.5 1.93 2.16 5.411
16S 205.4 1.98 2.12 4.109
cagA 430.2 1.87 2.23 8.604
ipaH 333.7 1.94 2.13 6.674

* ng/μl: DNA concentration
** A260/A280: bacteria concentration
*** A260/A230: DNA purity, a value between 1.8 and 2.0 are considered as normal
**** A260: RNA residue, a value approximate to 2 is normal

7.2.2. PCR of four DNA fragments (cagA/ipaH/invA/16S)

Reagents: sgDNA (16), oligoDNA (8)
Purpose: PCR the sgRNA and prove that the target bacterial has been successfully transformed with the plasmid
  1. Three different sets of forward and reverse primers will be used to test the plasmid sample. With a total of four plasmid samples, twelve 200μl PCR tubes will be used. Different primers are depicted in the graph below:
    Number of Primer(s)
    Integrated
    sequence
    sgDNA(1)*
    forward
    sgDNA(1)
    reverse
    sgDNA(2)
    forward
    sgDNA(2)
    reverse
    oligoDNA**
    forward
    oligoDNA
    reverse
    16S T7-F 16S(-)1 T7-F 16S(-)2 16S-F 16S-R
    cagA T7-F cagA(-)1 T7-F cagA(-)2 cagA-F cagA-R
    invA T7-F invA(-)1 T7-F invA(-)2 invA-F invA-R
    ipaH T7-F ipaH(-)1 T7-F ipaH(-)2 ipaH-F ipaH-R

    *Two sets of sgDNAs will be used for each plasmid sequence. However, both will use the same forward primer. This is to compare and contrast the efficiency of primers of different lengths.
    **oligo DNA: forward and reverse primers start at the 5’ end of the upper and the lower strand of the integrated sequence. This set is to prove that the viral DNA has been integrated successfully into the plasmid.

  2. Aliquot the following reagents and cultures into each of the twelve PCR tubes in their assigned volumes using a pipette.
    Total Volume 120 μl
    2× concentrated MasterMix 60μl
    ddH2O 42μl
    Primer-forward 6μl
    Primer-reverse 6μl
    Template(plasmid) 6μl

    (2 possible candidates for sgRNA in the form of DNA and 1 oligoDNA for each of the four strands will undergo amplification)

  3. Place the PCR tubes into the thermocycler and run the PCR (lasting for approximately an hour)

7.2.3. Gel electrophoresis of DNA

Purpose:
1. Check if PCR results are successful (when compared with original strands and the marker)
2. Separation of pure sgRNA and oligo DNA strips

Goal: In this experiment, we run the PCR results of the two target DNAs (that will be reverse-transcribed into sgRNA), oligo DNA, and the original plasmid (before PCR, as a control group) for each of the 4 groups. There will be a total of 31 wells.
Equipment:
1. 4 patches of gel with a total of 31 wells
2. Each well has a capacity of 60μl

  1. Gel electrophoresis preparation (Agarose TAE gel solution preparation)
    1. Add 1g of agarose* powder and 100ml of TAE** into a conical flask using a spatula and a measuring cylinder
    2. Heat until agarose powder fully dissolves
    3. Until the solution cools down to room temperature, add 10μl nucleic acid dye into the mixture. The solution turns clear pink.
    4. Place the combs on the buffer dams
    5. Pour the Agarose-TAE solution onto the casting tray
    6. The gel is allowed to solidify, leaving a gel slab with a roll of wells on one end
    7. Place the solidified gel into a chamber filled with TAE buffer

    *agarose: agarose creates a sieve that separates DNA with nuance length differences
    **TAE: TAE buffer provides ions during the electrophoresis. We prepared the 300ml of the solution by adding the following reagents together in the following ratio: TAE: ddH2O = 1:50

  2. Using a pipette, add the following mixtures in their directed portions into the wells
    1. Target DNA (will be used as the template for sgRNA):
      1. Each bacterial strain (a total of 4 strains: cagA, 16S, invA, ipaH) will occupy 4 wells, two for sgRNA1 and two for sgRNA 2. A total of 16 (4*4) wells will be used. The reagents in the four wells are shown below:
        ii. sgRNA template 1a (55 μl)
        iii. sgRNA template 1b (55 μl)
        iv. sgRNA template 2a (55 μl)
        v. sgRNA template 2b (55 μl)

        *sgRNA 1a and 1b are obtained from the same tube. The same is for 2a and 2b.

      2. Add one marker (20μl) in the first well of each gel to help identify the length of the target sequence.
      3. Total wells: 3 markers + 4 * 4 = 19 wells
    2. oligo DNA:
      1. Arrangement of each bacteria sample (a total of 4: cagA, 16S, invA, ipaH) on gel: original plasmid (10μl) + PCR oligo DNA 1 (55μl) + PCR oligo DNA 2 (40 μl*)
      2. One marker (20μl) in the first well of each gel strip to help identify the length of target sequence
      3. Total wells: 1 + 4 * 3 = 13 wells
  3. Conduct electrophoresis
    1. Each side of the “electrophoresis tray” is connected to a pole (the red wire is connected to the positive pole, while the black wire is connected to the negative pole)
    2. Run electrophoresis at 400mA, 140V for 20 minutes
    3. Observe results under a UV light and compare sample strips to the marker during electrophoresis. It’s possible to identify different samples through their different positions in the tray.
  4. Recovery of DNA from gels
    Purpose: Recover target DNA for later use
    1. Put the gel under UV light* and cut off the light banded agar, and put the pieces into separate microcentrifuge tubes, each with one target sequence
    2. Add in a 1:3 ratio of buffer B2 solution to dissolve the agar (compare to mass)

      Agar Mass (g) and volume of Buffer B2 (μl) added

      Oligo DNA SgRNA 1 SgRNA 2
      cagA 0.30g: 900 μl 0.670g: 250 μl (Actual) 0.408g: 1200 μl
      16S 0.4078: 1200 μl 0.311g: 900 μl 0.767g: 2000 μl
      invA 0.306g: 900 μl 0.327g: 1000 μl 0.623g: 1850 μl
      ipaH 0.201g: 1000 μl 0.423g: 1360 μl 0.542g: 1600 μl
    3. After the gel dissolved, heat it at 55°C in a water bath for 5-8 minutes
    4. Centrifuge the gel solution for 30s at 8000×g, dispose of filtrate in the bottom of the tubes
    5. Add in 300 μl Buffer B2, centrifuge for 30s at 8000×g
    6. Remove unwanted solution in the bottom tube, add in 500 μl Wash solution, centrifuge for 30s at 9000×g, then remove unwanted solution in the bottom tube
    7. Repeat steps 6 and 7.
    8. Centrifuge again without adding any reagents for 60s at 9000×g
    9. Leave the centrifuge tubes in a ventilated area for 5 minutes to allow the evaporation of ethanol
    10. Add 27 μl Elution Buffer into the tubes, then centrifuge 60s at 9000×g
    11. The solution at the bottom of each tube is the recycled DNA
  5. Test DNA concentration
    Purpose: Check if the DNA concentration is suitable for later usage and that sufficient DNAs are recovered

    Same procedure as “B. Test OD value of pET-28a integrated E. coli (dedicated to producing Cas12a protein)”, except set Elution Buffer as the control group before testing the concentration

    The test results are shown in the table below:

    cagA 16S invA ipaH
    Oligo DNAs 38.65 108.1 64.95 4.9
    SgRNA 1 12.75 0.4 20.75 28.4
    SgRNA 2 11.3 17.7 17.84 15.8

7.3.1. DNA in vitro transcription

Purpose: Transcribe the target DNA into sgRNA that can be utilized as a guilder in the final system
Apparatus: pipette , micro-centrifuge tube (size 0.2ml), clean bench, thermal cycler, test tube rack
Reagents: T7 transcription kit, T7 RNA polymerase, RNase Free Water, 75% Ethanol

  1. Prepare two micro-centrifuge tubes with size 0.2ml and put them into a cask
  2. Transfuse 10μl of T7 Transcription Kit into each of the tubes
  3. Check the concentration (ng/μl) of the template obtained in 7.2.4. and calculate an appropriate volume of template in which the mass of sgRNA should be equal or greater than 50ng.
  4. After identifying the volume needed that satisfies the mass requirement of sgRNA, in a total volume of 9μl, the RNase-free water needed will be 9μl subtract the volume the of template. Depending on the volume of the template and RNase-free water needed, the one with greater volume needs to be transfused into the tubes first, and then the other one.
  5. Add 1μl of T7 RNA Polymerase to all tubes, creating a system of 20μl. (If there are any solution droplets adhering to the tube walls, apply a few seconds of centrifugation) The content in each of the test tubes is recorded in the chart below.
    Name of Reagent/Template SgRNA1 cagA SgRNA2 cagA SgRNA1 ipaH SgRNA2 ipaH SgRNA1 invA SgRNA2 invA SgRNA1 16S SgRNA2 16S
    T7 Transcription Kit added(μl) 10 10 10 10 10 10 10 10
    T7 RNA Polymerase added(μl) 1 1 1 1 1 1 1 1
    RNase Free Water added(μl) 4 4 3 3 3 3 9 3
    Template added(μl) 5 5 6 6 6 6 0 6
  6. Place all tubes into the thermal cycler at 37°C for 2 hours.
  7. After incubation, use Nanodrop to measure the concentration of the product RNA; set DNA in vitro transcription reagent* as the controlled group and record the results.

Results are shown in the graph below:

Test result/Template SgRNA1 cagA SgRNA2 cagA SgRNA1 ipaH SgRNA2 ipaH SgRNA1 invA SgRNA2 invA SgRNA1 16S SgRNA2 16S
Concentration (ng/ μl) 3197.0 2871.3 2702.2 3116.0 3169.0 2368.7 3696.1 2759.1
A260/A280 1.97 1.96 1.95 1.99 1.98 1.97 2.00 1.93
A260/A230 2.34 2.33 2.27 2.39 2.37 2.27 2.37 2.16

* DNA in vitro transcription reagent group:

  • T7 Transcription Kit (10μl)
  • T7 RNA Polymerase (1μl)
  • RNase Free Water (9μl)

7.3.2. sgRNA purification

  1. In a 2ml microtube, add 350 μL Buffer RLT* using a pipette
  2. Add 80μL sgRNA sample into the same microtube
  3. Add in 250μL AR, and anhydrous ethanol, mix gently while also preparing the RNeasy spin column, and transfer all solutions into the column
  4. Add 350 μL Buffer RW1 to the RNeasy spin column. Close the lid, and centrifuge for 15s of 8000 g. Discard the flow through.
  5. Add 500 μL Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 15s of 12000 g. Discard the flow through.
  6. Add 500 μL Buffer RPE to the RNeasy spin column. Close the lid, and centrifuge for 2 min of 12000 g. Discard the flow through.
  7. Place the RNeasy spin column in a new 1.5ml collection tube. Add 30 μL RNase-free water directly to the spin column membrane. Close the lid, and centrifuge for 2 min of 8000g to elute the RNA.
  8. The flow through is the purified RNA.
  9. Test RNA concentration

    Same procedure as “B. Test OD value of pET-28a integrated E. coli (dedicated to produce Cas12a protein)”, except set ddH2O as the controlled group.

Test results:

ng/ μL A260/A280*
16S sgRNA 1 3.1 1.20
16S sgRNA 2 11.5 1.04
invA sgRNA 1 10.3 1.62
invA sgRNA 2 14.7 1.62
ipaH sgRNA 1 12.5 2.11
ipaH sgRNA 2 13.6 1.53
cagA sgRNA 1 461.5 1.04
cagA sgRNA 2 12.4 1.64

*A260/A280: a ratio near 1.60 depict a good result