Separating micro-organisms




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Two layers of liquid should form.


  1. Leave the tube for 2-3 minutes without disturbing it.




  1. Gently rock the liquids taking care not to let the two

layers mix too much.
RESULT
Describe the DNA which you have precipitated


DNA FROM CAVIAR
Source: online Bioscience journal

http://www.bioscience-explained.org/EN1.1/pdf/CaviarEN.pdf
AIM
This simple practical procedure allows the isolation of impure DNA from ‘caviar’ or fish eggs. The result is a pellet of thread-like material, which includes DNA but will still be contaminated with lipids, carbohydrates and proteins.
EQUIPMENT AND MATERIALS
Needed by each person or group


  • 20-30 g caviar (about 2 heaped teaspoonsful) e.g. roe from capelin (Mallotus villosus) or lumpsucker (Cyclopteris lumpus).


Note: such roe is sold under the Abba® brand name. The yellow or ‘natural’ variety works best.


  • 15 ml washing-up liquid e.g. Fairy Liquid, diluted 1:10 with distilled water




  • 1 teaspoon (about 6 g) of table salt




  • 2 ml ethanol. This must be ice-cold and at least 80% pure.




  • 3-4 drops of protease, e.g. Novozymes Neutrase ®




  • Glass rod







  • Funnel




  • Small test tube




  • Dropper or pipette for dispensing the enzyme




  • Pasteur pipette, the tip of which has been melted and curved to form a small hook

PROCEDURE


  1. Add the caviar and salt to a mortar, then crush the eggs using a pestle. The shells of the eggs have to be broken. Proteins are precipitated by the salt.




  1. Add the washing-up liquid solution to the mortar. The liquid should cover the caviar completely. The detergent dissolves lipids from the membranes of the roe.




  1. Add 3-4 drops of protease to the mixture and stir vigorously. The enzyme will partially degrade any soluble proteins.






  1. Filter the mixture through the coffee filter and collect the filtrate in a clean test tube.




  1. Add the ice-cold ethanol by carefully pouring it along the wall of the tube or use a pipette and add it at the bottom of the test tube. DNA precipitates as long threads in cold ethanol and can be found at the interface between the detergent solution and the ethanol.




  1. Collect the DNA with the help of a Pasteur pipette with a hooked tip. The DNA may be transferred to a microcentrifuge tube and stored, frozen, for later use e.g. for gel electrophoresis or staining of the DNA.




RESTRICTION ENZYME DIGESTS & DNA GEL ELECTROPHORESIS
Source: Adapted from NCBE ‘The Lambda DNA Protocol’ at http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PDF/LambdaSG.pdf
A technical/teachers guide to this activity can be found at http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PDF/LambdaTG.pdf



  1. CUTTING THE LAMBDA DNA WITH RESTRICTION ENZYMES

The restriction enzymes are in the following tubes:


Yellow – empty

Green – HindIII

Blue – Bam H1

Pink – EcoR1


Wear gloves (to protect the DNA from digestion by enzymes in your sweat).
Add a fresh tip to the Gilson pipette.

Practice aspirating and ejecting 10l of blue dye. [Volume errors in pipetting when using such small volumes can make big errors in your results].


Add a fresh tip to the pipette. Put 20 l of your chosen DNA solution into an enzyme tube of your choice.
Mix the liquid with the dried enzyme by carefully drawing the liquid up and down in the tip a few times. The liquid in the enzyme tube should have a distinct blue colour and there should be no concentration of dye at the bottom of the tube.
Cap the tube containing the enzyme and DNA, then incubate it at 37C in a waterbath for 30-45 minutes.
Repeat with the other enzyme tubes using a fresh tip each time. It helps to flick the tubes occasionally during incubation, to ensure that their contents are thoroughly mixed.


  1. LOADING THE GELS

After the incubation, add 2 l of loading dye to each enzyme tube using a fresh tip. [The loading dye contains sucrose so the sample will sink to the bottom of the well in the gel and not float away in the buffer in the tank. It also is blue so you can see your samples].


Mix the loading dye and the DNA sample very thoroughly by drawing the mixture up and down the pipette tip.
Put the gel and tank over a piece of black paper or card to make the wells more visible. Pour 10 ml of TBE buffer onto the gel. Load 10l of the mixture of loading dye and DNA into one of the wells, holding the tip above the well but under the buffer solution. Take great care not to puncture the bottom of the well with the pipette tip. Depress the pipette plunger gently and the DNA and dye mixture should sink into the well as it leaves the pipette tip.
Make a note of which DNA you have put in each well.

Repeat these steps with the other DNA samples using a fresh tip for each sample.


Load dye markers (5 l) in one well.


  1. RUNNING THE GEL

Fit a piece of carbon fibre tissue to each end of the tank so one end of it is in the buffer.


Check the power pack is turned OFF.
Put a lid on your tank.
Using the red and black leads, connect the tissue to the power source. The red lead should go to the positive and be at the end of the tank furthest from the wells. The black lead should go to the negative and be at the end nearest to the wells. [The reason it is this way round is that DNA in this running buffer, has a negative charge and will be attracted to the positive terminal (anode) when a current is applied].
After a few minutes you should see bubbles forming at the cathode (negative end of the tank). After a few more minutes, the DNA and dye mixture should start migrating through the gel. If it is going the wrong way, or not moving at all, you have connected your leads up incorrectly.

Try again.


The gel should run until the blue dye (which runs in front of any DNA fragments) reaches the end of the gel. This will probably take a few hours. Switch off the power and remove the leads. If you let it run too long, the DNA will come off the end of the gel and be lost in the buffer!!!


  1. STAINING THE GEL

Wear plastic gloves.

Pour off the buffer solution and pour the stain onto the surface of the gel.

Leave it for exactly 4 minutes, then return stain to the beaker/stain bottle.

Very carefully wash the gel surface with cold distilled water to remove excess stain. Repeat 3-4 times, finally pouring off all the water from the gel.

The remaining stain will gradually move down through the gel, staining the DNA as it does so. Faint bands should start to appear after 10 minutes.

Better results are seen if the gel is left to develop over night. Leave the gel in a plastic bag in the fridge, to prevent the gel from drying out.



-GALACTOSIDASE INDUCTION
Source: Adapted from NCBE Illuminating DNA

http://www.ncbe.reading.ac.uk/ncbe/PROTOCOLS/DNA/PDF/DNA06.pdf

The lac operon is the classic example of gene regulation, in which the production of -galactosidase (lactase) is induced by the presence of lactose in the growth medium. In this practical task, ONPG, rather than lactose, is used as a substrate for the enzyme.


Aim
To induce and measure the production of the enzyme -galactosidase (lactase) by E.coli.
Day 1: Preparation
You will need cultures of E.coli from a strain that possesses the lacZ (-galactosidase) gene. These can be grown on solid agar 24-48 hours in advance. To induce the production of -galactosidase, lactose must be present in the growth medium.

From stock E.coli plate prepare the following two streak plates:


E.coli on nutrient agar

E.coli on nutrient agar and lactose.
Incubate at 30C for 24-48 hours.
Day 2: Timing
This activity takes about 60 minutes, including an incubation period of 10 minutes.
Materials and equipment needed by each person or group


  • Cultures of E.coli

  • ONPG (ortho-nitrophenyl--D-galactoside) solution (2 cm3 per test sample)

  • Methylbenzene; 1 drop per test sample

  • Test tubes, caps, rack and marker pen

  • Inoculation loop

  • Pasteur pipettes

  • 5 cm3 syringe, for transferring ONPG solution

  • Waste container with disinfectant

  • Stopclock

  • Safety spectacles


Procedure
Quick qualitative method


  1. Using a syringe, transfer 2 cm3 of ONPG solution into each of two test tubes. One of these tubes will be a ‘control’; the other will be for the test culture.

  2. Label the tubes appropriately.

  3. Use a flamed wire loop to aseptically transfer a large colony of E.coli from the nutrient agar plate into the test solution. Suspend the microorganisms by agitating the loop, then sterilise it by flaming. Take care to introduce the loop into the flame slowly, to avoid sputtering! Repeat the process, this time transferring an E.coli colony from the plate where nutrient agar contains lactose, into the test solution.

  4. Add a drop of methylbenzene to each tube, cap the tubes and shake well to mix. Methylbenzene kills the cells and partially disrupts the cell membranes, allowing the ONPG to diffuse into the cells.

  5. Let the test tubes stand on the bench, until a strong yellow colour develops. This generally takes 5-20 minutes. The reaction can be speeded up by incubating the tubes at 37C. The colourless ONPG is broken down by -galactosidase to produce galactose and ortho-nitrophenyl (ONP). ONP is bright yellow in alkaline conditions.

  6. Compare the colour of the two tubes.



Safety
Handling microorganisms and methylbenzene


  • Good microbiological practice must be observed when handling microorganisms.



  • Methylbenzene is flammable and produces harmful vapour. Large volumes should therefore be handled in a fume cupboard, although the small amounts used here can safely be handled at the bench (but keep away from flames). Skin and eye contact should be avoided.




  • Eye protection must be worn.


Qualitative method
STEPS
1&2 Transfer 2cm3 of ONPG solution into each of two test tubes. Label the

tubes appropriately.



3. Aseptically transfer a colony from the plate to one of the tubes of ONPG solution. Twiddle to disperse the cells, then flame the loop to kill any remaining cells.


4-6. Add a drop of methylbenzene to each tube, cap the tubes and shake well to mix. Stand the tubes for 5-20 minutes until a yellow colour develops.



GENETIC CONTROL
Source: Adapted from NCBE Practical Biotechnology
http://www.ncbe.reading.ac.uk/NCBE/PROTOCOLS/PRACBIOTECH/PDF/catmilk.pdf

The genes found in living cells control the production of proteins such as enzymes. Any one cell can contain many thousands of genes all coding for different proteins. In a multicellular organism only a small proportion of the genes available within the cell will be required. Scientists think that genes become switched on or off as they are required. We are going to look at the gene which codes for an enzyme which breaks down the sugar LACTOSE. The, so called, Jacob-Monod hypothesis suggests that the gene which produces the enzyme required to break down lactose becomes switched on only when the substrate is present. When there is no lactose present the gene would be switched off.


Firstly you will set up an experiment to investigate the effect that the enzyme has on its substrate.
Production of Lactose – free Milk
The naturally occurring sugar found in milk is lactose. This disaccharide sugar is broken down to glucose and galactose by the enzyme  galactosidase. Many people (75% of the world’s adult population) cannot digest lactose because they cannot produce this enzyme. They are known as lactose intolerant. These people cannot consume milk or milk products.
In this experiment you are going to attempt to produce lactose free milk by immobilising the  galactosidase enzyme and setting up a continuous flow system.
APPARATUS
galactosidase enzyme

calcium chloride

sodium alginate

milk


2 x 10 ml syringe

250 ml beaker

2 x 100 ml beaker

circle of gauze

stirring rod

tea strainer

3 way tap

2 clinistix

paper towel
METHOD
Step 1 Immobilising the  galactosidase


  1. Use a 10 ml syringe to draw up 2 ml of  galactosidase enzyme




  1. Collect a piece of paper towel and then add 8 ml of sodium alginate to the same syringe (use the paper towel to catch any alginate drips)




  1. Rock the syringe until the two liquids mix completely (this may take about 5 minutes)




  1. Collect a 250 ml beaker and add 100 ml of calcium chloride


  1. Add the alginate/enzyme mixture to the calcium

chloride one drop at a time. Leave the beads in

the calcium chloride for 3 minutes to allow them

to set.



  1. Filter the immobilised enzyme beads from the

calcium chloride solution and then rinse the beads

in distilled water



Step 2 Setting up a Continuous Flow System




  1. Collect a 3 way tap and check that you know how

to use it. Practise with water.


  1. Place a piece of nylon gauze at the base of a

10 ml syringe and attach a 3 way tap to the end of

the syringe.




  1. Add the beads to the syringe.




  1. Collect 25 ml of milk in a beaker.



  1. Test the milk for the presence of glucose using a

CLINISTIX (Clinistix changes from pink to purple

if glucose is present).



  1. Slowly add the milk to the syringe and alter the 3 way tap to ensure that the milk passes through at a slow but steady rate. Collect the treated milk in a small beaker.




  1. Test the milk produced for the presence of glucose


RESULT:




Clinistix colour


Glucose presence

(+ or -)

Milk before enzyme treatment








Milk after enzyme treatment










CONCLUSION:

QUESTIONS:


  1. Name the enzyme which breaks down lactose ______________________




  1. What induces the production of this enzyme? ______________________

___________________________________________________________




  1. Give 2 advantages of using a continuous flow system, such as this, rather than a batch process to produce lactose free milk.

___________________________________________________________


___________________________________________________________


  1. Cheese making produces the waste product whey which is rich in lactose. The whey is usually dumped at sea. Describe a way in which this waste product might be upgraded into a useful product.

___________________________________________________________


___________________________________________________________
Lactose Metabolism in E. Coli
The bacterium E. coli produces  galactosidase only when lactose is present in it’s growing medium. The Jacob-Monod hypothesis suggests that the genes controlling the production of this enzyme work in the following way.
There are 3 genes involved:
The structural gene – codes for the enzyme
The operator gene – switches on the structural gene
The regulator gene – codes for a repressor molecule


DNA chain

Regulator gene




Operator gene

Structural gene

DNA chain




Repressor molecule

The presence of lactose induces the production of the enzyme. Lactose is therefore known as the inducer.


LACTOSE ABSENT
When lactose is absent the repressor molecule combines with the operator, the structural gene is switched OFF and no  galacosidase is produced.


DNA chain

Regulator gene




Operator gene

Structural gene

OFF


DNA chain




repressor



LACTOSE PRESENT
When lactose is present some of it binds with the repressor and the operator gene is able to switch ON the structural gene and  galactosidase is produced.


DNA chain

Regulator gene




Operator gene

Structural gene

ON


DNA chain




repressor

lactose

When all the lactose has been digested the repressor molecule will bind again with the operator and the gene will be switched back off.



QUESTIONS


  1. Name the 3 genes involved with this hypothesis ____________________

___________________________________________________________




  1. Which gene codes for the enzyme? ______________________________

___________________________________________________________




  1. What does the repressor molecule do? ___________________________

___________________________________________________________




  1. Why is lactose known as the inducer? ____________________________

___________________________________________________________




  1. Why might this system of gene control be described as energy-efficient?

___________________________________________________________


___________________________________________________________


  1. Complete the blanks in the following passage.

In E. coli a _________________________ gene codes for the production


of an enzyme called ____________________. When lactose is absent a
___________________ molecule binds with the ___________________
gene and the structural gene is switched ____________________ .
ANTIBODY/ANTIGEN REACTIONS

Source: James Watt College
Suppliers of antibodies (antiserum) and antigens include Alba Bioscience (formerly the Scottish Antibody Production Unit) http://www.show.scot.nhs.uk/sapu/index.html and Sigma-Aldrich http://www.sigmaaldrich.com/Area_of_Interest/Europe_Home/UK.html

This simple experiment utilizes (purified) agar plates with wells cut into them as depicted here:



Antibody

Distance between central well & test wells must be equal



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