Lab 4: Microbial Genetics

Gene Expression

• Genes are expressed constantly throughout a cell's life, others are needed based on growth phase or environment
• Cells conserve energy by making proteins only when needed

The Operon Model

• Proposed to explain why Escherichia coli produces β-galactosidase when lactose is present, but not when it is absent
• An operon includes a promoter, an operator, and structural genes
• The promoter is where RNA polymerase binds
• The operator signals whether transcription will occur
• Structural genes code for peptides
• A regulatory gene encodes a regulatory protein that binds to the operator to activate or inhibit transcription
• In an inducible operon, transcription is activated by a substance such as lactose
• The regulatory protein has an allosteric site that binds an inducer, inactivating the repressor
• A repressible operon is inhibited when a specific small molecule is present
• Tryptophan inhibits its own synthesis in E. coli
• If tryptophan binds to the regulatory protein, the complex represses further tryptophan synthesis

nar Operon and ara Operon

• In E. coli, nitrate reductase genes are encoded by the nar operon
• The regulatory gene (narXL) encodes the regulatory protein narL
• Enzymes for arabinose catabolism are encoded by the ara operon
• The regulatory gene (araC) encodes the regulatory protein araC
• Regulatory genes for these operons produce proteins that bind to the operator, either inhibiting transcription or promoting RNA polymerase binding, depending on the shape of the regulatory proteins

Regulatory Proteins

• Regulatory proteins that control the nar and ara operons can either inhibit or activate transcription
• Regulatory proteins bind to the operator and prevent transcription
• A change in the shape of regulatory proteins activates transcription of the structural genes
• Ara structural genes were replaced with pGLO which encodes green fluorescent protein (gfp)
• The pGLO gene, sourced from the jellyfish Aequoria victoria, researches gene expression
• pGLO is a trademark of Bio-Rad Laboratories
• E. coli colonies fluoresce under UV light due to the jellyfish gene for GFP

Investigative Exercise: Nitrate Reductase

• Label two small tubes "Aerobic" and "Anaerobic," add 0.5 ml of E. coli to each, then 0.2 ml nitrate solution
• Place the "anaerobic" tube in an anaerobic Brewer jar and add the activated charcoal sachet
• Shake the "aerobic" tube to aerate the culture
• After 15 minutes, test for nitrite ions by adding 5 drops of nitrate reagent A and 5 drops of nitrate reagent B to each tube.
• A pink color indicates nitrite ions

Investigative Exercise: Arabinose Catabolism

• Transfer 0.1 ml of E. coli pGLO culture to glucose-nutrient agar, glucose-arabinose-nutrient agar, and arabinose-nutrient agar
• Disinfect a spreading rod by dipping it in alcohol and igniting it
• While the alcohol burns off, hold the spreading rod pointed down
• Spread the liquid on each plate's surface
• Incubate the plates inverted at 35°C for 24-48 hours

Metabolic Mutants

• Genes and their characteristics are generally stable, but genetic variants can appear due to mutations
• A mutation is a change in the sequence of nucleotide bases in the cell's DNA
• Catabolic mutations result in a bacterium unable to produce an enzyme needed to utilize a substrate
• Anabolic mutations result in bacteria unable to synthesize an essential organic chemical
• Wild-type bacteria are called prototrophs
• Mutants are called auxotrophs; some cannot catabolize certain organic substrates or synthesize organic chemicals
• Cultures can synthesize growth requirements from glucose-minimal salts medium
• Mutations are induced by exposing cells to UV light; auxotrophs unable to grow on glucose-minimal salts medium will be identified

Replica Plating

• Due to the low rate of recognizable mutants, special techniques can select for desired mutants
• Direct selection picks out mutant cells while rejecting unmutated parent cells
• Indirect selection will be used with the replica plating technique in this exercise
• Mutated bacteria are grown on a nutritionally complete solid medium
• Auxotrophs grow on the complete medium but not on the minimal medium
• An imprint of the colonies is made on velveteen-covered or rubber-coated blocks, transferred to a glucose-minimal salts solid medium and a complete solid medium

Culture - Serratia marcescens

• Requires spreading rod technique (Exercise 27), Pipetting (Appendix A) and serial dilution technique (Appendix B)

Investigative Exercise

• Label nutrient agar plates “A,” “B,” and “C” and dilution blanks “1,” “2,” and “3.”
• Aseptically pipette 1 ml of Serratia into dilution blank 1 and mix well.
• Transfer 1 ml from dilution blank 1 to dilution blank 2, and mix well Pour 1 ml from dilution blank 2 to bottle 3 and 1 ml to the surface of plate A.
• Mix bottle 3, transfer 1 ml to the surface of plate B with a sterile pipette and 0.1 ml to the surface of plate C.
• Disinfect a spreading rod by dipping it in alcohol, quickly igniting the alcohol in a Bunsen burner flame and letting the alcohol burn off and cool.
• Spread the liquid on the surface of plate C over the entire surface and Do the same with plates B and A.
• Put the plates with the covers off, about 30 cm from the ultraviolet lamp. Position the plates directly under the lamp. Turn the lamp on for 30 to 60 seconds

Procedure Second Period

• Select a plate with 25 to 50 isolated colonies - this is the master plate.
• Mark the uninoculated complete and minimal media with a reference mark on the bottom of each plate.
• Carefully open the package of velveteen by placing the replicator block on the center of the velveteen, pick up the four corners of the cloth, and secure them tightly on the handle with a rubber band.
• Inoculate the sterile media using either step a or step b, by holding the replica-plating block by resting it on the table with the rough surface
• Invert the master plate selected in step 1 on the block, and allow the master plate agar to lightly touch the block.
• Remove the lid from the minimal medium, align the reference marks, and touch the uninoculated minimal agar with the inoculated replica-plating block.
• Place the reference marks in a 12 o’clock position, and remove the lids. Touch the replica-plating block to the master plate; then, without altering its orientation, gently touch it to the minimal medium, then to the complete medium (Figure 28.2C). Replace the lids, and incubate, then refrigerate the master plate..

Procedure Third Period

• Compare the plates after incubation and Record your results

Isolation of DNA

• Transformation is a rare event where a recipient bacterium acquires small DNA pieces from a dead donor
• These acquired DNA pieces can give the recipient bacterium new traits
• Escherichia coli bacteria will be transformed with a gene encoding green fluorescent protein (GFP)
• E. coli K-12, is used because of long history of safe commercial use,
• E. coli K-12 does not normally colonize the human intestine and survives poorly in the environment
• GFP causes the jellyfish to fluoresce under ultraviolet light
• To genetically modify a bacterium, the desired gene is put into a plasmid
• The pGLO plasmid has been genetically modified to carry the gfp and ampicillin-resistance genes
• The ampicillin-resistance gene enables direct selection of transformed cells
• E. coli bacteria are not naturally competent and don't readily take up foreign DNA
• Treatment with Ca2+ neutralizes negatively charged phosphates on DNA and the phospholipid membrane
• Heat shocking increases plasma membrane fluidity, allowing DNA to enter
• LB agar, similar to nutrient agar, is used and is nutritionally rich to grow E. coli K-12; it contains 0.1% tryptose and 0.5% yeast extract

Preparing Competent Cells

• Using a sterile loop, transfer two to four large colonies of E. coli to one tube, then Mix with the loop, until the entire colony is dispersed in the CaCl2 solution and place the tube back on ice
• Aseptically pipette of pGLO plasmid into the tube and mix. Incubate the tubes in ice for 10 minutes.
• Place both tubes in a foam floating rack and Float both tubes in a 42°C water bath for exactly 50 seconds then quickly move the tubes to ice and Incubate the tubes in ice for 2 minutes.
• Aseptically add of LB broth to each tube. Incubate the tubes for 10 minutes at room temperature.
• Gently flick the tubes to resuspend the bacteria and Using a different pipette tip for each tube, transfer from each tube onto the appropriate nutrient agar plate.
• Spread cells evenly over one plate with the sterile spreading rod

Procedure Third Period

• Do not look at the ultraviolet light, and do not leave your hand exposed to it.
• Wearing safety goggles, examine your plates in visible light and under UV light. Count the colonies, and record your results.