Bios1034_practical_123-march 2019_final.docx

Module Code: BIOS1034 Applied Genetics

DRS SANDY LOH / MS KAN

BIOS1034 Applied Genetics Practical Classes 2019 Background and Safety Introduction for Experiments 1-3 This set of practical classes is intended to introduce you to some of the practical techniques used to study the genetics of prokaryotic organisms and to illustrate some of the concepts introduced in lectures. For this purpose, you will be using the bacterium Escherichia coli K12 (commonly referred to as E. coli). E. coli may have been studied more thoroughly at the genetic level than any other organism. This is because it is easy to handle (and will grow on a growth medium consisting of simple inorganic salts and a sugar), it is haploid, it has a short generation time (20 minutes under optimal conditions for strain K12), it has one very small circular chromosome (four and a half million nucleotide pairs) and despite the fact that E. coli has no true sexual process, it is possible to perform genetic exchange experiments quite readily. The microorganisms that you are using are classified as ACDP category 1 (the least hazardous). However, you should remember that E. coli is a human gut commensal, some strains of which are serious pathogens, although the lab strains that you will be using have been attenuated by decades of lab culture. Ingestion of the 'phages may also temporarily kill some of your normal gut flora, allowing other bacteria to thrive in the lack of competition and this may also cause mild gastroenteritis. Ensure that you employ aseptic technique at all times for your own good and to prevent contamination of your cultures. Be careful what you do with contaminated items and ensure that there is always disinfectant readily available to mop up spills. IF IN DOUBT ABOUT THE CORRECT PROCEDURE, ASK A DEMONSTRATOR BEFORE YOU COMMENCE WORK.

As in any laboratory, you are not permitted to eat, drink, chew gum, smoke, apply cosmetics or do anything else that may cause potentially contaminated fingers to come into contact with your mouth and you must wear a lab coat and safety glasses at all times.

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Module Code: BIOS1034 Applied Genetics

DRS SANDY LOH / MS KAN

Experiment 1 Selection of Spontaneous Mutants The purpose of this experiment is to illustrate the occurrence of spontaneous mutants in a population and the usefulness of appropriate selective procedures. Wild-type E. coli K12 is unable to grow in the presence of the amino acid valine except when the amino acid isoleucine is also present. However, spontaneous mutants arise which have mutations in the genes that control the isoleucine and valine biosynthesis operon, and these are able to grow in the presence of valine alone. DAY 1 (13 Mar) 1. You are supplied with a culture of strain W grown in M9 minimal medium (containing simple inorganic salts and sugar) and two M9 minimal agar plates without isoleucine, one of which contains amino acid valine and one of which does not. 2. Label the agar plate with your initials and ASEPTICALLY pipette 0.1 ml (100 l) of the culture onto the surface of the agar. 3. Use a sterile spreader to spread the bacterial suspension as evenly as possible over the surface of the agar. 4. Put the plate to one side to allow the liquid to be absorbed into the agar gel. 5. When the surface of the agar is dry, turn the plate upside down and place it in the tray provided. These plates will be incubated at 37C for one day and observed in the next practical session. DAY 2 (15 Mar) 6. Examine your plates and record which plate(s) each colony has grown on. 7. Have you identified any valine-resistant mutants? What fraction of the original culture were these? Assume that a stationary liquid culture of E. coli contains 2.5  109 bacterial cells ml–1.

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Module Code: BIOS1034 Applied Genetics

DRS SANDY LOH / MS KAN

Experiment 2 Bacteriophage Growth and Titration Bacteriophage lambda propagates itself by attaching to the cell surface and injecting its DNA into the cell. This replicates up hundreds of times within the cell and is packaged into new 'phage particles. The cell is then lysed, releasing the new 'phage particles to infect perhaps fifty other surrounding cells. The progeny 'phages from these infections in turn infect several hundred other cells. If bacterial cells are seeded onto a plate with a few 'phage particles, then the bacteria will grow to form a turbid (cloudy) lawn of bacteria. However, where a 'phage particle landed, all of the bacteria in the vicinity will have been killed to produce a clear disk one or two millimetres in diameter called a plaque. DAY 1 (13 Mar) 1. You are supplied with a culture of phages. Dilute phages with SM buffer at 100-1, 1002 , 100-3, 100-4, 100-5. 2. Add 10 l of diluted phages into 100 l of bacterial stock. 3. Incubate the mixture at 37oC for 20 min (this step is particularly important for phages to attach to bacterial cells). 4. Add the mixture into 3 ml soft molten agar. 5. Mix the mixture rapidly but steadily. Pour onto a pre-labelled LB agar plate to cover the overall agar surface and allow the molten agar to solidify (10-15 min). 6. Incubate the plate at 37oC overnight. DAY 2 (15 Mar) 7. The biological activity of your virus and is expressed as plaque forming units (pfu) per ml. Observe and count the phage plaques (only plate with 10-100 plaques is reliable for counting). Then, use the formula given below to calculate the viral titer. Formula, pfu/ml =

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑞𝑢𝑒𝑠 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑋 𝑣𝑜𝑙𝑢𝑚𝑒

Where, Dilution = the dilution of the plate that you counted Volume = volume of diluted phage added into bacteria in ml (0.01 ml)

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Module Code: BIOS1034 Applied Genetics

DRS SANDY LOH / MS KAN

Experiment 3 GFP expression in E. coli Arabinose Operon mutant The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms (GMOs). The plasmid contains several reporter genes, most notably for the green fluorescent protein (GFP) and ampicillin resistance gene. GFP gene was isolated from the jellyfish Aequorea victoria. Because it shares a bidirectional promoter with a gene for metabolizing arabinose, the GFP gene is expressed in the presence of arabinose, which makes the transgenic organism expressing its fluorescence detected under UV light. GFP can be induced in bacteria containing the pGLO plasmid by growing them on agar plates supplemented with arabinose. Basically, the GFP signal is proportional to the concentration of arabinose fed. DAY 1 (13 Mar) Experiment 3.1: 1. You are provided with different LB agar plates: a) 1 LB agar + Ampicillin + Arabinose (0.2% w/v) b) 1 LB agar + Ampicillin c) 1 LB agar + Arabinose (0.2% w/v) 2. Use aseptic method to take a loop-full of bacterial culture and streak on each agar plate. 3. Incubate all agar plates at 37°C for overnight. DAY 2 (15 Mar) Experiment 3.1: 4.

Observe and record any bacterial growth on the agar plates under UV light.

Experiment 3.2: 1. You are provided 5 flasks of bacterial culture which have been induced for three hours with different concentrations of arabinose – 0.01%, 0.05%, 0.1%, 0.2%, 0.5% (w/v) by which flasks are randomly labelled. 2. Observe the level of GFP protein expression under the UV light. 3. Arrange the bacterial culture flask according to the ascending level of expression from low to high. 4

Module Code: BIOS1034 Applied Genetics

DRS SANDY LOH / MS KAN

4. Record the label sequence in the table below. Arabinose (w/v)

0.01%

0.05%

Flask label

5

0.1%

0.02%

0.5%