Micro 329 Lecture 8

• Bacterial Transformation occurred on 2/19/25

Lab Pointers

• Move LB broth to the fridge.
• Spread IPTG and X-gal first.
• Never vortex competent E. coli.
• Never centrifuge competent E. coli.
• Recovery media lacks ampicillin; only LB plates and LB broth for overnight growth contain ampicillin.
• Cells require recovery post-heat shock to avoid being killed by ampicillin, as they need time to produce ampicillin resistance protein.
• Limit plate incubation to 18 hours.
• Throwing away transformation mixes or ligation mixes is not recommended.
• Maintain a timely pace without rushing.

Controls

• A: Negative blank with undigested plasmid
• B: Positive control with undigested plasmid
• C: Recircularization control, digested, and dephosphorylated plasmid.
• D-G: Different insert to vector ligation ratios.

Draft Introduction

• Ensure a draft is written of the introduction
• Constructive criticism on the draft will be strictly graded.
• A draft review form located on Canvas should be used.

Steps

• Isolate plasmid DNA from E. coli.
• Isolate chromosomal DNA from Aliivibrio fischeri.
• Cut the DNA into fragments.
• Ligate fragments into a cloning vector.
• Transform the vector into E. coli.
• Induce the expression of the lac operon in the vector.
• Screen for bioluminescence.

History

• Transformation was first demonstrated in 1928 by Frederick Griffiths.
• Avirulent Streptococcus pneumoniae grew in the presence of heat-killed virulent S. pneumoniae.
• The virulence gene(s) are transferable.
• In 1944 Avery, MacCleod, and McCarty showed experimentally that purified DNA from virulent strains was the transforming principle.

Bacterial Competence

• Bacterial competence means the ability of a cell to uptake extracellular DNA from its surrounding environment.

Natural vs Artificial Competence

• Natural competence involves using DNA as nutrients and promoting genetic diversity, also adapting to adverse environmental conditions.
• Examples of naturally competent species include Bacillus subtilis, Pseudomonas aeruginosa, and Streptococcus pneumoniae.
• Artificial competence is commonly used in molecular biology.
• E. coli is not naturally competent
• E. coli needs to be artificially made competent.

Artificial Competence: Methods

• Chemical competence employs a classic method.
• Cells are resuspended in ice-cold CaCl₂.
• Cells are then heat-shocked at 42°C for a precise duration.
• The action operates through an unknown mechanism.
• Chemical competence is less efficient but cheaper than electroporation.
• Electro competence uses electroporation.
• Applies an electric current that briefly opens trans-membrane pores, allowing DNA to enter cells.
• The action causes membrane damage, and it requires cells to repair themselves prior to growth.

Penicillins

• Penicillins, like Ampicillin, originate from a fungal source.
• All penicillins are based on a B-lactam ring and a thiazolidine ring.
• Lactam refers to a cyclic amide group.
• Penicillins target the cell wall, preventing cross-linking of peptidoglycan.
• Many bacteria produce resistant B-lactamases.
• The drug is effective against G+ cocci and some G- cocci, along with spirochetes.

Blue-White Screening

• Blue-white screening serves as an easy method to determine if a transformed colony contains a plasmid with an insert.
• Also known as a-complementation.
• The screening uses the lacZ gene (Beta-Galactosidase).
• White colonies indicate an insert = success.
• Blue colonies indicate no insert = failure.

How Blue/White Screening Works

• Wild-type E. coli contains the lacZ gene.
• This gene encodes B-galactosidase for lactose utilization as a carbon/energy source.
• Can also degrade X-gal.
• IPTG induces LacZ production.
• X-gal is converted to 5-5-dibromo-4,4-dichloro-indigo.
• Oxidized 5-5-dibromo-4,4-dichloro-indigo is dark blue.
• The alpha domain of LacZ is missing in the E. coli being used.
• A functional LacZ not produced by the E. coli unless the plasmid is present.
• Adding a plasmid with a mutated LacZ alpha domain results in non-functional colonies.
• Successfully ligating the Lux operon into the LacZ locus yields white colonies.
• When the vector recirularizes, a functional LacZ alpha domain results in blue colonies.
• This is in the presence of X-gal and IPTG, which is undesired.

pGEM Plasmid

• The multiple cloning site (MCS) is located inside of the lacZ gene
• The gene's function is interrupted when an insert is present at the MCS.