Microbiology and Bacterial Transformation Notes

Gelatin Hydrolysis

• Gelatin is hydrolyzed by the exoenzyme gelatinase.
• Nutrient gelatin dissolves in warm water ($50^oC$), solidifies (gels) when cooled below $20^oC$, and liquefies (sols) when heated.
• When gelatin is hydrolyzed by gelatinase, it liquefies and does not solidify even when cooled below $20^oC$.
  • Solidifies (gels): (-) result
  • Liquefies (sols): (+) result

Urea Hydrolysis

• Urea is a waste product of protein digestion, excreted in urine.
• Urease liberates ammonia from urea: $Urea + H<em>2O \xrightarrow{Urease} 2NH</em>3 + CO_2$
• Urea broth contains peptone, glucose, urea, and phenol red.
  • Uninoculated broth: pH 6.8 (salmon color)
  • Positive result: pH 8.4 (fuchsia, hot pink)

Amino Acids Catabolism: Deamination

• Deamination is the removal of an amino group.
  • The amino group is converted into ammonia, which can be excreted from the cell.
  • Results in organic acid production.
• Phenylalanine Deamination can be detected by:
  • Adding 4-5 drops of 10% ferric chloride solution to phenylalanine slant: a dark green color indicates a ferric ion complex with the organic acid.
  • Nessler's reagent: deep yellow indicates the presence of ammonia.
• $Phenylalanine \xrightarrow{Phenylalanine deaminase} Phenylpyruvic acid + NH_3$
• $Phenylpyruvic acid + Ferric ion (Fe^{3+}) \rightarrow Green complex$
• $NH_3 + Nessler’s reagent \rightarrow Yellow$

Amino Acids Catabolism: Decarboxylation

• Decarboxylation is the removal of carbon dioxide from an amino acid.
• The presence of a specific decarboxylase enzyme results in the breakdown of the amino acid with the formation of the corresponding amine, liberation of $CO_2$, and a shift in pH to alkaline.
• $Ornithine \xrightarrow{Ornithine decarboxylase} Putrescine$
• pH indicator: Bromcresol purple
  • Positive result: Lavender-purple
  • Negative result: Yellow

Indole Production

• Some bacteria can convert the amino acid tryptophan to indole: the enzyme tryptophanase can convert tryptophan to indole, ammonia, and pyruvic acid.
• $Tryptophan \xrightarrow{Tryptophanase} Indole + Pyruvic acid + NH_3$
• Indole production can be detected by adding 4-5 drops of Kovac’s reagent to MIO medium.
• The formation of a red ring on the surface of the medium indicates a (+) result.

Sulfur Reduction ($H_2S$ production)

• Some bacteria can liberate hydrogen sulfide ($H_2S$) from the sulfur-containing amino acids: cystine, cysteine, and methionine.
• $H_2S$ can also be produced from the reduction of inorganic compounds such as thiosulfate.
• $H_2S$ production can be detected by adding a heavy metal salt such as iron or lead to the culture medium.
• When $H_2S$ is produced, sulfide reacts with the metal salt to produce a visible black precipitate.

Bacterial Transformation

• Bacteria are prokaryotes: single-celled organisms without a nucleus.
• Cell structures include:
  • Cell membrane
  • Cell wall
  • Chromosome
  • Plasmid
  • Flagellum
  • Pilus
  • Capsule

Bacterial Reproduction

• Bacteria reproduce asexually using binary fission, creating perfectly identical copies.
• They divide at a geometric rate.

Bacterial Conjugation

• Bacteria reproduce sexually using conjugation.
• Bacteria exchange plasmid DNA.
• This is how bacteria become antibiotic-resistant.
• The rate at which they can share plasmids is staggering, allowing us to:
  • Add DNA foreign to the bacteria and exploit the reproduction rate for our own benefit.

Antibiotic Resistance

• Antibiotics are chemicals that normally kill bacteria (such as penicillin).
• We may add a gene to a bacterium that will make it resistant to antibiotics (in other words, antibiotics will no longer kill it).
• The antibiotic used in this lab is called ampicillin (amp).
• If we put ampicillin into LB broth, most bacteria will die, but bacteria carrying the amp-resistance gene will survive.

pGLO Plasmid

• Components of the pGLO plasmid:
  • Beta Lactamase (bla):
    • Ampicillin resistance that is always on.
  • GFP (Green Fluorescent Protein):
    • Jellyfish gene that produces fluorescent glow under UV light.
  • araC regulator protein:
    • Regulates transcription of GFP.
    • Works only in the presence of arabinose (a sugar).

Green Fluorescent Protein (GFP)

• In this lab, we will be inserting a gene that codes for the protein GFP.
• GFP stands for Green Fluorescent Protein.
• This gene was extracted from a jellyfish that fluoresces naturally.

Arabinose Operon

• Three genes (araB, araA, and araD) code for three enzymes involved in the breakdown of arabinose, clustered together in the arabinose operon.
• Arabinose interacts directly with araC, causing it to change its shape; this promotes the binding of RNA polymerase, and the three genes araB, araA, and araD are transcribed.
• The three enzymes produced break down arabinose.
• In the absence of arabinose, the araC returns to its original shape, and transcription is shut off.
• The DNA code of the pGLO plasmid has been engineered to incorporate aspects of the arabinose operon.
• Both the promoter (PBAD) and the araC gene are present; however, the genes that code for arabinose catabolism (araB, A, and D) have been replaced by the single gene that codes for GFP.
• Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase, and GFP is produced.
• Cells fluoresce brilliant green as they produce more and more GFP.
• In the absence of arabinose, the GFP gene is not transcribed.

pGLO Transformation Process

• Overview:
  • Foreign DNA (region of interest, GFP) is inserted into a pGLO plasmid.
  • The recombinant DNA (pGLO plasmid with GFP) is introduced into a bacteria cell.
  • The cell is plated on a medium + antibiotic.
  • Only bacteria containing the recombinant DNA grow and culture.

Solutions

• Luria-Bertani (LB) broth:
  • Medium that contains nutrients for bacterial growth and gene expression.
  • Contains carbohydrates, amino acids, nucleotides, salts, and vitamins.
• Transformation Solution (Calcium Chloride):
  • Neutralizes the repulsive negative charges of the phosphate backbone of the DNA and the phospholipids of the cell membrane.
  • Allows the DNA to enter the cells.

Experimental Procedure: Transformation Setup

• Label one closed tube "+pGLO" and another "-pGLO," and label both tubes with your group’s name.
• Place them in the foam tube rack.
• Open the tubes, and using a sterile transfer pipet, transfer 250 μl of transformation solution (CaCl2) into each tube.
• Place the tubes in the ice cup.

Adding Bacteria

• Use a sterile loop to pick up 5 colonies of bacteria from your starter plate.
• Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube.
• Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks).
• Place the tube back in the tube rack in the ice cup.
• Using a new sterile loop, repeat for the -pGLO tube.

Adding Plasmid DNA

• Instructor adds 10 μl of pGLO plasmid DNA to the +pGLO tube only.
• The pGLO plasmid is not added to the -pGLO tube (control).
• Both tubes go back into the ice cup.

Plating

• Label 4 plates on the bottom (not the lid) along the periphery:
  • +PGLO LB/amp (red stripe)
  • +PGLO LB/amp/ARA (green stripe)
  • -PGLO LB (black stripe)
  • -pGLO LB/amp (red stripe)

Heat Shock

• Heat shocking the bacteria with a sudden rise in temperature will help it take up foreign DNA.
• Using the foam rack, place the (+) pGLO and (-) pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds.
• Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water.
• When the 50 seconds are done, place the tubes back in the ice cup.
• For best transformation results, the change from the ice (0°C) to 42°C and then back to the ice must be rapid.
• Incubate tubes on ice for 2 minutes.

Incubation

• Remove the rack containing the tubes from the ice and place it on the bench top.
• Open a tube and, using a new sterile pipette, add 250 μl of LB broth to the + tube and reclose it.
• Repeat with a new sterile pipette for the - tube.
• Flick, tap to mix.
• Incubate the tubes for 10 minutes at 37°C.
• Tap the closed tubes with your finger to mix, then incubate the tubes for 30 minutes at 37°C.

Spreading and Streaking

• Remove the rack containing the tubes from the 37°C water bath.
• Tap the closed tubes with your finger to mix.
• Using a new sterile pipette for each tube, pipette 100 μl of the experiment and control onto the appropriate plates.
• Use a new sterile loop for each plate.
• Spread the suspensions evenly around the surface of the agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface or streak.

Final Steps

• Stack up your plates upside down and tape them together using the tape on the front bench.
• Put your group name on the stack and leave it on the front bench