Biology 107L: Labs 6, 7, and 8 - Microbial Genetics and Biotechnology

Vocabulary practice flashcards defining essential concepts, techniques, reagents, and mechanisms from Labs 6, 7, and 8, covering bacterial genetic exchange, plasmid biology, transformation efficiency, PCR, and gel electrophoresis.

Wild Type (wt)

The typical form of an organism, strain, or gene as it occurs in nature without mutations, such as the Acinetobacter calcoaceticus strain ADP-1.

Mutant Strain ADP-6

A genetically altered strain of Acinetobacter calcoaceticus that has mutations preventing the metabolism of para-hydroxybenzoate ($\text{pHB}$), allowing it to grow exclusively on succinate as its carbon source.

Para-hydroxybenzoate (pHB)

A carbon source that wild-type Acinetobacter ADP-1 can utilize for growth, but mutant ADP-6 cannot metabolize unless successfully transformed with wild-type DNA.

Natural Transformation

A naturally occurring process in which competent bacteria take up naked exogenous DNA directly from their surroundings and integrate it into their genome.

Bacterial Competence

The physiological state enabling a bacterial cell to take up free extracellular DNA; it can be induced by secreted signaling molecules, competence factors, or environmental stressors such as starvation.

Unnatural Transformation

An artificial laboratory procedure where exogenous DNA is forced into non-competent bacterial cells using physical or chemical methods such as heat shock or electroporation.

Transduction

A mode of horizontal gene transfer in bacteria where a bacteriophage (bacterial virus) mistakenly packages host bacterial DNA into its capsid and injects it into a recipient cell.

Bacterial Conjugation

The transfer of genetic material between bacterial cells through direct physical cell-to-cell contact mediated by a sex pilus.

F Factor (Fertility Factor)

A plasmid carrying genes that encode sex pilus proteins and mediate horizontal DNA transfer, converting recipient ($\text{F}^-$) cells into donor ($\text{F}^+$) cells.

Lysis Buffer

A specialized reagent used to break open bacterial cells and liberate genomic DNA, whose activity is visually confirmed by increasing solution cloudiness during incubation at $60^\circ\text{C}$.

Supernatant

The liquid phase positioned above a compacted pellet following centrifugation, which is decanted or discarded without disturbing the cell pellet.

Plasmid

A small, typically circular, double-stranded extrachromosomal DNA molecule in the cytoplasm of bacteria that replicates autonomously from the genomic chromosome.

Origin of Replication (ori)

A specific sequence motif on a plasmid required for recruiting host cell replication machinery to initiate plasmid copying.

Multiple Cloning Site (MCS)

A defined region within an engineered plasmid vector engineered with several unique restriction enzyme cleavage sites for inserting target genes of interest.

Selectable Marker

A plasmid-borne gene that imparts a survival advantage under specific culture conditions, such as conferring antibiotic resistance or permitting growth in nutrient-deficient media.

Ampicillin

A $\beta$-lactam antibiotic that interferes with bacterial cell wall synthesis during cellular replication, inhibiting bacterial growth.

$\beta$-lactamase

An enzyme that breaks down the structural core of ampicillin, deactivating the drug and enabling bacteria harboring the resistance gene to grow on ampicillin-supplemented media.

Green Fluorescent Protein (GFP)

A visual reporter protein originating from jellyfish, engineered for heightened brightness, that emits green light under ultraviolet (UV) illumination.

TNTC (Too Numerous To Count)

A plate-counting designation applied when colony growth is confluent (forming a bacterial lawn) or so dense that individual colonies cannot be accurately counted.

Colony Forming Unit (CFU)

A single viable bacterial cell or group of cells that divides and gives rise to a visible, macroscopic clonal colony on solid media.

Transformation Efficiency

The quantitative measure of the number of bacterial transformants (colonies) produced per microgram ($\mu\text{g}$) of plasmid DNA added during transformation.

Polymerase Chain Reaction (PCR)

An in vitro molecular technique used to exponentially replicate and amplify a defined DNA target sequence through automated thermal cycling.

Denaturation (PCR)

The first phase of a PCR cycle where thermal energy at $94^\circ\text{C}$ melts double-stranded template DNA apart into two single strands.

Annealing (PCR)

The second phase of a PCR cycle where reaction temperatures are reduced to approximately $55^\circ\text{C}$, allowing short oligonucleotide primers to bind specifically to complementary sequences on template strands.

Extension (PCR)

The third phase of a PCR cycle where temperature is raised to $72^\circ\text{C}$, optimizing Taq DNA polymerase activity to synthesize the nascent complementary strand using free dNTPs.

Taq DNA Polymerase

A heat-resistant DNA polymerase enzyme capable of synthesizing new DNA strands complementary to single-stranded templates at elevated temperatures during PCR.

Deoxynucleoside Triphosphates (dNTPs)

Free nucleotide monomers (dATP, dCTP, dGTP, and dTTP) that supply both the requisite nucleotides and the chemical energy needed for DNA chain elongation.

Oligonucleotide

A short, single-stranded synthetic sequence of DNA nucleotides utilized in PCR primer pairs or as overlapping building blocks during synthetic gene synthesis.

Antiparallel (DNA)

The structural property of a double-stranded DNA molecule wherein one strand runs in the $5'\rightarrow 3'$ orientation while the complementary partner strand runs in the $3'\rightarrow 5'$ orientation.

Codon

A specific sequence of three adjacent DNA or RNA nucleotides that encodes an individual amino acid or signals the termination of protein translation.

Gel Electrophoresis

A laboratory method that separates negatively charged nucleic acid fragments through an agarose gel matrix toward a positive electrode, with smaller fragments traveling faster and farther than larger fragments.

DNA Ladder

A reference standard containing a series of DNA fragments of predetermined base-pair sizes, used in gel electrophoresis to estimate the size of unknown DNA bands.

Succinate

A primary carbon source that both wild-type Acinetobacter ADP-1 and mutant ADP-6 can utilize to sustain growth on minimal media.

SSC Buffer (Saline-Sodium Citrate)

A specialized salt solution used in transformation protocols to resuspend bacterial pellets and carry out tenfold serial dilutions of cell lysates.

Touchdown PCR

A modified thermal cycling protocol where the annealing temperature is incrementally lowered across initial cycles to favor specific primer binding and minimize non-specific amplification.

Thermal Cycler (Thermocycler)

An automated laboratory instrument engineered to rapidly alter block temperatures through precise denaturation, annealing, and extension phases.

Loading Dye

A tracking solution containing dense reagents to sink DNA samples into agarose wells and colored indicators to monitor migration during electrophoresis.

Complementary Base Pairing

The structural bonding rule governing double-stranded nucleic acids, where adenine pairs with thymine via two hydrogen bonds and cytosine pairs with guanine via three hydrogen bonds.

DNA Phosphate Backbone Charge

The recurring chemical property that imparts a net negative charge to nucleic acid molecules, causing them to migrate toward the positive anode during gel electrophoresis.

Negative Control Plate

An agar plate—such as mutant ADP-6 plated without wild-type lysate—used to verify the absence of contamination, revertant colonies, or surviving unlysed cells.

Synthetic Gene Assembly

A molecular method using thermal cycling to anneal and elongate overlapping single-stranded oligonucleotides into a single contiguous $180\,\text{bp}$ construct.

Transformation Efficiency Calculation

$\text{TE} = \left(\frac{\text{Number of Colonies}}{\mu\text{g of DNA}}\right) \times \left(\frac{\text{Total Cell Volume}}{\text{Plated Cell Volume}}\right)$, expressed in units of $\text{CFU}/\mu\text{g}$.

Expected growth on the mutant ADP-6 control plate on $\text{pHB}$ media and the biological rationale

Zero colonies, because mutant ADP-6 cells lack the functional metabolic genes necessary to utilize para-hydroxybenzoate as a sole carbon source.

Expected growth on the undiluted wild-type lysate control plate and the rationale

Zero colonies, because detergent incubation at $60^\circ\text{C}$ lyses and destroys all viable donor cells, leaving only free non-viable extracellular DNA.

Expected result of plating recipient ADP-6 cells mixed with wild-type lysate on $\text{pHB}$ agar

Colony growth, because competent mutant cells naturally take up wild-type genomic fragments and integrate functional $\text{pHB}$-metabolizing genes through homologous recombination.

Expected colony count trend across $10^{-3}$ through $10^{-6}$ lysate dilutions

A successive tenfold decrease in colony formation, because serial dilution reduces the concentration of available wild-type transforming DNA.

Expected growth outcome for non-transformed bacteria plated on ampicillin-supplemented media

No growth, because ampicillin prevents bacterial cell wall synthesis during replication unless cleaved by plasmid-encoded $\beta$-lactamase.

Expected phenotype of transformed colonies harboring the GFP plasmid under ultraviolet (UV) illumination

Green fluorescence, because the plasmid encodes an engineered jellyfish reporter protein that emits green light upon UV excitation.

Expected phenotype of colonies transformed with a non-GFP control plasmid on ampicillin agar under UV light

White or cream non-fluorescent colonies, because the plasmid provides $\beta$-lactamase for survival but lacks the gene encoding the fluorescent reporter protein.

Expected band size for the experimental synthetic gene PCR product on an agarose gel

A single distinct band at $180\,\text{bp}$, resulting from Taq polymerase successfully extending and joining the six overlapping single-stranded oligonucleotides.

Expected gel electrophoresis result for the PCR negative control lacking Taq polymerase

Absence of the $180\,\text{bp}$ target band, because chain elongation and fragment assembly require enzymatic synthesis by DNA polymerase.

Expected gel migration pattern of small DNA fragments compared to large DNA fragments

Smaller fragments migrate farther toward the positive anode than larger fragments because they experience less friction moving through the porous agarose matrix.