Team:Hong Kong UCCKE/Assays


Assay

In order to test the presence and effectiveness of the transformed E.coli containing BBa_K3077100, we carried out two assays. Firstly, we verified the presence of the DNA. Secondly, we tested the catalytic activity on Tributyrin Agar of the bacteria. We also transformed the genes of BBa_K3077000 and BBa_K3077200 into E.coli. Unfortunately, we don’t have enough time to test their effectiveness as planned, so will be continued in our future project.


Assay 1: DNA verification

Aim:

To determine the presence or absence of insert DNA (composite part BBa_K3077100) in 6 plasmid constructs

Method:

Colony PCR followed by gel electrophoresis

Colony PCR

Colony PCR mixture: (per PCR tube)

Components Colonies
DNA
Prefix forward 0.5
VR 0.5
Taq 0.6
Thermal pol 2.5
MQ 20.4

Protocol: (Referenced from NEB Taq polymerase PCR Protocol [1])

  1. Prepare the PCR mixture by assembling all the components above.
  2. Gently mix the mixture
  3. Suspend a colony into the PCR mixture in PCR tube. Streak the same colony on a different plate to save a copy of the colony. Do the same for the 6 colonies.
  4. Put the PCR tubes into the thermal cycler and perform the following steps
    5. Step Temperature Time
    Initial Denaturation 95°C 30 seconds
    Cycles x25 95°C
    68°C (annealing temperature)     		30 seconds
    1 minute/kb
    Final Extension 68°C 5 minutes

  5. Hold the PCR tubes at 8°C

Gel Electrophoresis

Ingredients:

Components Amount
Agarose powder 0.6 g
TAE Buffer 180 ml
DNA Ladder 10 µl
Green Dye SYBR 6 µl
Loading Buffer (for each clone) 2 µl
PCR mixture from Colony PCR (of each clone) 10 µl

Protocol:

Prepare Agarose Gel

  1. Measure 0.6 g Agarose powder and add it to a 200 ml conical flask.
  2. Add 60 ml TAE Buffer to the flask to make 1% agarose gel.
  3. Cover the opening with aluminium foil.
  4. Melt the agarose using the hot plate until the solution becomes clear.
  5. Let the solution cool to about 50-55°C using water bath, swirl the flask occasionally to cool evenly.
  6. Add 6 ul of SYBR Green dye into the solution. (SYBR Green is light-sensitive, keep it dark).
  7. Seal the ends of the casting tray with two layers of tap.
  8. Place the combs in the gel casting tray.
  9. Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white).
  10. Carefully pull out the combs and remove the tape.
  11. Place the gel in the electrophoresis chamber.
  12. Add enough TAE Buffer (About 120 ml) so that there is about 2-3 mm of buffer over the gel.

Loading The Gel

  1. Record the order to add each clone.
  2. Add 2 ul loading buffer on parafilm.
  3. Carefully pipette 10 ul of each clone and mix with loading buffer.
  4. Load the mixed sample to the corresponding well.
  5. Pipette 10 ul of the DNA ladder marker into at least one well of each row on the gel.

Running The Gel

  1. Place the lid on the gel box, connecting the electrodes.
  2. Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)
  3. Turn on the power supply to 100 volts.
  4. Check to make sure the current is running through the buffer by looking for bubbles forming on each electrode.
  5. Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the DNA).
  6. Let the power run for 30 minutes.
  7. Record the results using gel documentation system.
  8. Turn off the power.
  9. Disconnect the wires from the power supply.
  10. Remove the lid of the electrophoresis chamber.
  11. Using gloves, carefully remove the tray and gel.

Reference:

[1]: PCR Protocol for Taq DNA Polymerase with Standard Taq Buffer (M0273) from NEB (https://international.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273)


Assay 2: Lipid Hydrolysis Assay


Aim

To test the catalytic activity of the composite part BBa_K3077100 we designed with Tributyrin agar.

Materials

Ingredients Amount
Special peptone 5.0g
Yeast extract 3.0g
Agar 12.0g
Distilled water 1L
Tributyrin (neutral)(91010) 10g


Protocol

  1. Prepare a mixture of 5g Special peptone, 3g Yeast extract and 12g Agar.
  2. Storage of prepared media: below 8°C, protected it from direct light.
    Storage of dehydrated powder: in a dry place, in tightly-sealed containers at 2-25°C.
  3. Dissolve 20 g of prepared media, in 1 litre of distilled water.
  4. Sterilize it by autoclaving at 121°C for 15 minutes.
  5. Let cool to 80°C and add 10g neutral Tributyrin (91010).
  6. Mix thoroughly to emulsify the Tributyrin completely. Pour plates in order to maintain uniform turbidity.
  7. Spread E.coli transformed with the composite part BBa_K3077100 plasmid on the Tributyrin agar plate.
  8. Observe and record the clear zone around the E.coli after incubating the cells for 1 day.

Principles

Tributyrin Agar was originally formulated by Anderson [1] for the detection and enumeration of lipolytic microorganisms such as Staphylococci [2], Clostridia [3], marine Flavobacteria and Pseudomonas [4] and moulds in foodstuffs and other materials. Special peptone and yeast extract provide nutrients e.g. organic nitrogen, carbon compounds and other growth factors like vitamins to the organisms. Tributyrin degradation by the lipase is indicated by clear zones surrounding the E.coli in the otherwise turbid culture medium.

References:

[1]: J.A. Anderson, Ber. Iird. Int. Mikrobiol. Kongress, 3, 726-728 (1939)

[2]: A.G. Imnes, J. Appl. Bact., 19, 39-45 (1956)

[3]: A.T. Willis, J. Path. Bact., 80, 379-390 (1960)

[4]: P.R. Hayes, J. Gen. Microbiol., 30, 1-19 (1963)

[5]: This protocol is referenced from Millipore 91015 Tributyrin Agar (https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma-Aldrich/Datasheet/1/91015dat.pdf)