Lab 5: Molecular Biology and Microbiology Techniques

Lab 5: Growth of Microbes (part 2) and DNA digestion

Lab 5 Learning Outcomes

Digest plasmid DNA using restriction enzymes.
Analyze, discuss, and interpret results from microbial plating experiments.
Load an agarose gel with a digested DNA sample.
Construct restriction maps for linear and circular DNA molecules.

Part A: Restriction Digestion of Plasmid DNA

Introduction

Restriction enzymes are part of the bacterial defense system against viruses and can be used to cut DNA at specific sites.
This allows scientists to build recombinant DNA molecules.
It's a useful diagnostic tool to 'map' how various DNA fragments fit together in one molecule.
In this exercise, you will work with a recombinant plasmid (called 46-15) made for use in zebra fish to study toxicity of heavy metal ions in water.
The aim was to combine the region of a gene (gfp) from a jellyfish that codes for a green fluorescent protein, with the promoter and regulatory region (p) from the zebra fish metallothionein (MT) gene whose expression is induced by heavy metals.
After the recombinant pMT-gfp DNA is transformed into zebra fish, the gfp gene should be expressed in the presence of heavy metals and the fish should fluoresce.
The task is to test that the recombinant plasmid containing the MT promoter (pMT) and gfp coding region has been constructed correctly before it is transformed into the fish using restriction enzymes as a diagnostic tool.
The DNA fragments will be separated by size using agarose gel electrophoresis, enabling one to work out the sizes of the DNA fragments and construct a plasmid restriction map.
Restriction enzymes are more correctly termed restriction endonucleases.
- Restriction means to cut or digest.
- Endo refers to the inside of the molecule (as opposed to the ends of the molecule).
- Nuclease refers to the digestion of nucleic acid such as DNA.
Commonly used restriction enzymes recognize a palindromic (inverted repeat) sequence in double-stranded DNA and then cleave the sugar-phosphate backbone of the DNA at specific sites.
In this experiment, you will use restriction enzymes EcoRI and HindIII, which have the following recognition sites:
- EcoRI: 5’ – GAATTC – 3’ 3’ – CTTAAG – 5’
- HindIII: 5’ – AAGCTT – 3’ 3’ – TTCGAA – 5’
  *The arrows indicate the precise position at which each DNA strand is cut/digested.
The weak hydrogen bonds joining the two strands between the cutting sites will be broken, and the DNA fragments will fall apart.
Restriction enzymes are named for the bacterial species from which they were derived: Escherichia coli in the case of EcoRI and Haemophilus influenzae in the case of HindIII.
Restriction enzymes require a buffer with essential salts, including magnesium ions.
Each person must set up one restriction digest reaction individually (as shown in Table 5.3).
After one hour of incubation, the digested DNA will be ready to load on the agarose gel, and electrophoresis carried out.

Materials (per four students, each student sets up one reaction)

Gel-loading dye
DNA of Plasmid 46-15 (P)
Restriction Buffer (B)
Distilled water (sterile)
1. 5 ml microcentrifuge tubes
Restriction enzymes EcoRI (E) and HindIII (H) MUST BE KEPT COLD (in ice bucket)
Pipette (p20)
Yellow pipette tips
Water baths at 37oC & polystyrene floats

Method

It's important to pipette extremely small volumes with accuracy and precision.
Too much buffer will result in the salt concentration being wrong, and restriction enzymes may not work properly.
Too little of the restriction enzyme, the DNA will not digest completely.

Practice dispensing small volumes using a pipette:
- Using the gel-loading dye, practice taking up and ejecting small volumes with the p20 pipette. This pipette will accurately measure 2-20 $μl$ volumes. Eject the liquid onto a piece of parafilm.
- First dispense 20 $μl$ , then dispense 10 $μl$ and finally dispense 2 $μl$ . Observe the level of liquid in the yellow tip for each of the different volumes and continue to practice until you are confident that you can accurately dispense small volumes of solution.
In groups of four, work out which digest you will set up:
- The plasmid DNA will be digested with each enzyme separately, or both enzymes together, or no enzyme (control experiment).
- In groups of four, there are four restriction digests to set up (shown in Table 5.3). Each person should set up one of the digests (A to D).
- Label a clean, empty 1.5 ml microcentrifuge tube on the lid and on the side using the black marker pen provided. You must include: Bench # Digest # Your initials

Individually set up your restriction digest:

Remember to use a new clean pipette tip for each reagent to avoid contamination. BE ESPECIALLY CAREFUL WITH THE RESTRICTION ENZYMES: AVOID CONTAMINATION AND KEEP TUBES ON ICE AS MUCH AS POSSIBLE.
Set up your restriction digest in your labeled 1.5 ml microcentrifuge tube. Add the reagents to the bottom of the microcentrifuge tube in the order shown in Table 5.1, with water first and enzyme last. The order is important to ensure the correct buffer environment before adding the enzyme.
Tick each box in Table 5.1 after adding each reagent. That way if you get distracted, you will still know which reagents you have already added.

Table 5.1: VOLUME OF REAGENTS ( $\mu$ L) IN EACH RESTRICTION DIGEST.

Reagents	A No enzyme (control)	B EcoRI	C HindIII	D EcoRI + HindIII
Distilled H2O	16 $μl$	14 $μl$	14 $μl$	12 $μl$
Buffer	2 $μl$	2 $μl$	2 $μl$	2 $μl$
Plasmid DNA	2 $μl$	2 $μl$	2 $μl$	2 $μl$
EcoRI Add last!	-	2 $μl$	-	2 $μl$
HindIII	-	-	2 $μl$	2 $μl$
Total volume	20 $μl$	20 $μl$	20 $μl$	20 $μl$

Mix the reagents thoroughly and incubate:
- Once all the reagents have been added to the tube, close the lid and mix the contents by flicking the tube with your finger for several seconds. Thorough mixing is crucial for the success of your restriction digest. Restriction enzymes are supplied in a dense solution of 50% glycerol (to keep them liquid at –20oC where they are stored) and will sink to the bottom of your tube if not mixed properly.
- Pool all the reagents at the bottom of your tube by spinning briefly in a microfuge, using the ‘pulse’ spin button.
- Place your restriction digest (with the lid closed) in a floating rack placed in a water bath set at 37oC.
- Leave the reaction to incubate for one hour. Whilst you are waiting, proceed with parts B-D and the practice-gel loading section of Part E.

Part B: Sampling microbes from the environment

Describing Colony Appearance

Describing the appearance (morphology) of colonies is an important tool used by microbiologists to identify different types of microorganisms.
Some colonies may be colored, some circular in shape, while others are irregular.
The characteristics of a colony are termed the "colony morphology", and some of the terms used to describe these are:
- Circular
- Filamentous
- Irregular
- Rhizoid
- Spindle

Results

Work individually to analyze your results.
Observe the growth on each of your plates.
It may be difficult to make detailed observations because the plates may be crowded with growth and are sealed with Sellotape.
Do not open them, simply make whatever observations you can.

Try to identify each different type of colony on the plates and describe it using the terms found in the introduction, including the color. Determine the approximate number of each type of colony. If there are too many to count, record the information as TMTC.
When you have made your observations, dispose of the plates in the decontamination bin, then wash your hands thoroughly.

Table 5.2: Results of environmental sampling (swabbed plate)

Environment Sampled:
Diameter	Appearance

Table 5.3: Results of the plate exposed to air

Part C: Streaking plates with E.coli

Record results from streaking agar plate

Draw a diagram of your streaked plate. There should be the greatest number of cells in your first streaked area (A) and they should have grown into a dense confluent layer. Your second streaked region (B) may also be confluent. However, the number of cells should decrease from one area to the next. Somewhere on the plate (probably in section D), single cells should have been separated sufficiently to grow into single discrete colonies.

Self-assessment criteria:

A good number of well-separated colonies are formed on the streaked lines, and there are no obvious signs of faulty technique.
No overlap exists between the first and fourth streak.
The culture has been streaked as per instructions.
The plate is correctly labeled with your initials & family name, lab stream and seat bench location and what was streaked on the plate.

Part D: Testing the 5 second rule for food contamination

Draw your results, describe what you saw, and count colonies.

Results & Discussion

Do these results fit with your prediction? Yes/No/Inconclusive, because …
Is your hypothesis supported or not supported? If your hypothesis is supported, what further research could be conducted to obtain additional support for this hypothesis? If your hypothesis is not supported, what modified or alternative hypothesis do you propose?
Were there any limitations in your experimental design? If so, how would you improve it? You should consider your use of controls, how many variables you actually tested in your experiment, your use of replicates, possibility of cross-contamination, etc.
In retrospect, could you have stated a clearer hypothesis and prediction last week? If so, how would you restate these?

Part E: Gel Electrophoresis of Plasmid DNA

Principles

The DNA fragments from your restriction digest can be separated according to their size, using agarose gel electrophoresis.
This method utilizes the property of DNA being negatively charged, due to the phosphate group of each nucleotide in a DNA molecule.
Your restriction digest will be loaded into a well of an agarose gel, where an electrical current will be passed through the gel matrix (electrophoresis).
The electrical current enables the movement of DNA fragments towards the positive electrode (anode).
Smaller DNA fragments will move through the gel more quickly than larger DNA fragments, after electrophoresis DNA fragments will be separated according to their size.

Materials

Practice loading gels & solutions (a few per lab)
Agarose gel (0.8% agarose in 1x TBE buffer)
DNA size ladder (1 kb Plus DNA ladder)
Gel loading dye
Gel running buffer (1x TBE)
Gel boxes and loading sheets
*Power supply packs

Electrical Hazard:

Power Supplies for Gel Electrophoresis
*Students are not permitted to turn on a power supply without assistance from a demonstrator or technician.

Method

Individually practice loading an agarose gel
- Practice loading gels are available in the laboratory for you to use.
- As in the ‘real’ gel, you will see that each gel has holes (wells) in it, in which to load your sample.
- The wells do not go all the way down to the bottom of the gel; if they did your sample would leak out of the well underneath the gel and be lost in the buffer.
- Practice loading 20 $μl$ of the test solution into a well. Repeat until you are confident with loading samples into the wells. You need to load your sample so all of it goes in the well, being careful not to pierce the gel at the bottom of the well with your pipette tip.
Load your digested plasmid DNA samples into a well of an agarose gel
- After one hour of incubating your restriction digest at 37oC (from Part A).
- Add 2 $μl$ loading dye to your restriction digest reaction and mix thoroughly as before.
- The loading dye is a mixture of several reagents that will:
  - Stop the restriction endonuclease (enzyme) reaction.
  - Increase the density of your sample (so the sample will sink to the bottom of the well).
  - Allow you to visualise the movement of your sample through the agarose gel as electrophoresis proceeds.
- Working together in groups of four, locate the gel-loading sheet next to the gel you will use to load your samples. Fill in the required details on the gel-loading sheet with your bench number and digest number, ensuring each member of your group has room to load their restriction digests alongside one another on the same gel. Leave one spare lane for the DNA size marker.
- Each tube will now contain 22 $μl$ of solution. Load 20 $μl$ from each tube into the correct well of your assigned agarose gel. Dispense this volume slowly to the first stop on the pipette to avoid introducing air bubbles into the well.
- When all samples have been loaded, a demonstrator will load the DNA size ladder (1 kb Plus DNA ladder) that contains a variety of double-stranded DNA fragments of known sizes.
- The electrical supply will be connected, and the gels will be run at 100 volts for about 2 hours. You do not need to wait for the gel electrophoresis to be completed before leaving the laboratory.
- When the gel has run the technician will switch off the electrical current and remove the gel from the gel box. The DNA will be stained with a fluorescent dye, ethidium bromide, which allows the DNA to be visualised under UV light. Due to the hazardous nature of both ethidium bromide and UV irradiation, you will not be required to assist in this procedure.
- The technician will produce a photograph of your gel for you to analyse in the next lab session.

Concept Questions

Using a new micropipette tip for each reagent is important to avoid contamination between reagents.
DNA will move from the negative to the positive electrode because DNA is negatively charged due to the phosphate groups.
Since all DNA molecules are negatively charged, the size of the DNA fragment is most important in determining the rate of migration through the gel.
In gel electrophoresis, the marker DNA (e.g., the 1 Kb Plus DNA ladder used in this lab) is useful because it provides a reference for determining the sizes of the DNA fragments in the sample.

Part F: Restriction Mapping

Introduction

The sizes of DNA fragments used in restriction mapping are usually measured in nucleotide base-pairs (bp) or thousands of base-pairs (kilobase pairs or kbp).
- Kilobase pairs are commonly called ‘kilobases’ or ‘kb’ for short.
- Thus a DNA fragment of 6 kb is 6,000 base pairs long and a fragment of 1.5 kb is 1,500 base pairs long.
- Since restriction enzymes only cut double-stranded DNA, we assume that these 6 kb and 1.5 kb fragments are both double stranded.
Building a restriction map is like doing a jigsaw puzzle. There are several pieces that you have to fit together.
*Unlike a normal jigsaw puzzle, each restriction enzyme cuts the same DNA molecule up in a different way.
By working through the examples and problems, you will see how these restriction maps are fitted together.
Examples of restriction mapping for both CIRCULAR and LINEAR molecules are shown

Restriction Mapping Example 1 - CIRCULAR PLASMID DNA

The figure shows a representative agarose gel with separated DNA fragments that were obtained when the circular DNA plasmid pCELL-1 was digested with restriction enzymes EcoRI and BamHI.
The plasmid fragment sizes are determined by comparison to the DNA ladder, which consists of fragments of known sizes.
Smaller sized fragments will appear to be fainter than the larger fragments on the gel.

What is the total size of the plasmid?

The total size can be calculated by adding up the sizes of the fragments in any of the columns.
- For example, digestion with EcoRI gives one fragment of 10.0 kb, which is the plasmid size.
- This can be checked by adding up the fragment sizes in the BamHI or EcoRI & BamHI columns, which also each add up to 10 kb.

How many EcoRI recognition sites are there in plasmid pCELL-1?

Since one fragment is produced and the plasmid is a circular molecule, EcoRI must have one recognition site.

How many BamHI recognition sites are there in plasmid pCELL-1?

Since two fragments are produced and the plasmid is a circular molecule, BamHI must have two recognition sites.

Digestion with both EcoRI & BamHI

In the double-digest (EcoRI & BamHI), there are three fragments, as expected because EcoRI cuts once and BamHI cuts twice.
*The figure on the previous page that shows the sizes of DNA fragments suggests that the 7.0 kb BamHI fragment has been cut into two by EcoRI in the double digest. How can you tell this?
In the EcoRI & BamHI digest a fragment the same size as 3.0 kb BamHI fragment is still seen. However, instead of a 7.0 kb BamHI fragment there are fragments of 6.0 kb and 1.0 kb. This suggests the 7.0 kb fragment has been cut by EcoRI.

How does the whole map fit together?

The map can be drawn in two ways, which are both correct views of the same plasmid.

Restriction Mapping Example 2 - LINEAR DNA

Construct a restriction map of a LINEAR DNA plasmid using the following data:
- Your map should indicate the relative positions of the restriction sites along with distances between restriction sites:
- DNA Sizes of linear fragments (bp)
  1. DNA cut with BglII 300, 125
  2. DNA cut with HpaI 250, 100, 75
  3. DNA cut with BglII + HpaI 250, 100, 50, 25
    a) Start by working out how many restriction sites there are for each of the enzymes. (How many times would you have to cut a linear piece of string to get that number of fragments?)
BglII: 1
HpaI: 2
b) Draw the position of the single BglII cutting site on the restriction map and label the sizes of the two fragments: c) Compare digests 2 and 3 - which one of the three HpaI fragments has been cut by adding BglII to the mix? (Which of the 3 fragments from digest 2 have not been cut, and which one is now in two smaller pieces in digest 3?)
The 75 bp fragment is cut into 50 bp and 25 bp fragments by BglII.
d) Draw the two HpaI sites either side of the BglII site to make the 75 bp and 25 bp fragments.
Which one of the following is correct?
e) Write in all the fragment sizes to complete your restriction map. Make sure the sizes are all correct by comparing them to those in the question. Note you do not need to add restriction sites to the ends of linear molecules (they are already 'cut').
A mirror image of this map is also correct

Restriction Mapping Questions

Question 1

Construct a restriction map of a CIRCULAR DNA plasmid using the following data. Your map should indicate the relative positions of the restriction sites along with distances between restriction sites:

DNA Sizes of linear fragments (bp)
- DNA cut with BglII 7950
- DNA cut with HpaI 7950
- DNA cut with BglII + HpaI 6630, 1320
  Clue: work out the following first: How many times does BglII cut the DNA? How many times does HpaI cut the DNA? Do they cut at the same place?

Question 2

Construct a restriction map of a LINEAR fragment of DNA using the following data. Your map should indicate the relative positions of the restriction sites along with distances from the ends of the molecule to the restriction sites and between restriction sites.

Draw your final answer on the rectangle shown below.
DNA Sizes of Fragments (kb)
- DNA cut with EcoRI 8, 2
- DNA cut with SalI 5, 5
- DNA cut with EcoRI + SalI 5, 3, 2
  Work out the following first: How many times does EcoRI cut the DNA? How many times does SalI cut the DNA? Do they cut at the same place? How do the single and double digests differ?

Question 3

DNA Sizes of linear fragments (kb)
- DNA cut with EcoRI 5, 9
- DNA cut with SalI 3, 11
- DNA cut with EcoRI + SalI 1, 2, 4, 7
  Work out the following first: How many times does EcoRI cut the DNA? How many times does SalI cut the DNA? Do they cut at the same place?

Question 4

DNA cut with PstI 3.5, 12
DNA cut with BamHI 2, 4, 9.5
DNA cut with PstI + BamHI 1.5, 2, 4, 8
Work out the following first: How many times does PstI cut the DNA? How many times does BamHI cut the DNA? Do they cut at the same place?

Lab 5: Growth of Microbes (Part 2) and DNA Digestion Achievements Checklist

Practical skill achievements

Count bacterial colonies on agar plates and evaluate the results.
Digest plasmid DNA using restriction enzymes.
Load an agarose gel with a DNA sample.

Lab theory achievements

Determine if your hypothesis is supported or rejected by your experimental results.
Evaluate whether your experimental design or techniques could have been improved.
Construct restriction maps for linear and circular DNA molecules.

Other important lab work achievements

Clean and tidy bench and wash hands.

Preparation for Lab 6: Make sure you have attended or watched Lecture 26 (pedigrees, PCR and DNA profiling), as well as reading your lab manual, before coming to the lab.