BIO 120.11 | M4 Genetic Engineering

Genetic engineering is also known as _

Gene modification

Uses recombinant DNA (rDNA) and lab-based technologies to alter the genetic makeup of an organism

Genetic engineering (modification)

Enumerate 3 fields through which genetic engineering is usually applied

iam

• Industrial epfatb

  • Enzymes, plastics, food, additives, textiles, bioenergy

• Agriculture mps

  • Modified food, pest control, stress resistance

• Medical avt

  • Antibiotics, vaccines, therapeutics

3 other terms for Genetically Engineered Organisms (GEOs)

rgt

• Recombinant

• Genetically modified organisms (GMO)

• Transgenic

Other ways through which genetic engineering can be done apart from recombinant DNA

mcat

• Mutagenesis

• CRISPR/Cas9

• Allelic exchange

• Transposons

Earliest step for developing genetically modified organisms (GMOs)

Gene cloning

Explain 4 general steps in gene cloning

ilts

1. Isolation of the gene to be cloned

2. Ligation of the DNA or gene-of-interest into a vector

3. Transformation of host cell with recombinant DNA

4. Selection or screening of host cell with recombinant DNA

Host carrying the recombinant DNA

Recombinant organism

Contains foreign gene / gene-of-interest

Recombinant DNA

T/F: Any organism can be a source of recombinant DNA as long as the sequence of GOI is known

TRUE

3 methods through which isolation of gene to be cloned can be done

rpa

• Restriction enzymes

• PCR

• Artificial gene synthesis

Molecular scissors that accurately and reproducibly cut genomic DNA into fragments called _

• Restriction enzymes

• Restriction fragments

Has unique recognition sequences; gene (to be cloned/isolated) must be near or between restriction sites

Restriction enzymes

4 examples of restriction enzymes & source microbes

habs

(cohesive ends)

• HindIII = Haemophilus influenzae Rd

• AluI = Arthrobacter luteus

• BamHI = Bacillus amyloliquefaciens H

  (blunt end)

• Sau3AI = Staphylococcus aureus 3A

Polymerase Chain Reaction (PCR) is a rapid method of _

isolating and copying a gene

Can amplify specific DNA sequences that are present in very small quantities even when they are mixed with many other DNA molecules

PCR

3 steps in PCR amplification

dae

1. Denature DNA (92 - 95C)

2. Anneal primers (45 - 65C)

3. Extend primers (65 - 75C)

_-nucleases are used for restriction enzymes

Endo

If you have a linear DNA with 2 restriction sites, how many restriction fragments will be produced after introduction of RE?

3 RFs

If you have a circular DNA with 2 restriction sites, how many restriction fragments will be produced after introduction of RE?

2 RFs

During PCR denaturation, what type of bonds are broken?

Hydrogen bonds

T/F: In PCR amplification, DNA copies increase exponentially

TRUE

T/F: Artificial gene synthesis is also highly reliant on PCR

TRUE

Enumerate, explain 2 ways through which artificial gene synthesis can be done

1. Polymerase Cycling Assembly (PCA)

   1. Fragment preparation: GOI is cut into smaller fragments with overlapping regions to allow annealing during PCR

   2. PCR: Fragments are amplified in a series of PCR cycles, allowing overlapping regions between fragments to fuse in the process

   3. Assembly: As cycles progress, fragments join together in stepwise fashion, forming full-length genome

2. Ligation

   1. Short synthetic oligonucleotides are chemically synthesized based on desired genetic sequence

   2. Oligonucleotides are then assembled into larger fragments, which are then ligated together to form full-length genome

   3. Oligonucleotides synthesized are designed to have overlapping regions that can anneal to each other

T/F: DNA ligase creates covalent bonds between sugar and phosphate of adjacent nucleotides

TRUE

What type of bond is formed by ligase?

Phosphodiester bond

Stage where DNA/GOI is inserted into vector

Ligation of DNA into vector

5 kinds of vectors used for DNA ligation into vector

pbcyb

• Plasmids

• Bacteriophages

• Cosmids

• Yeast Artificial Chromosomes (YACs)

• Bacterial Artificial Chromosomes (BACs)

Vector consisting of cos site of phage + plasmid

Cosmid

Carriers of GOI into host cell; make numerous copies of the gene

Cloning vector

Enumerate parts of cloning vector

ossr

1. Origin of replication (ori)

   1. Ensures that vector replicates in the host cell

2. Several restriction enzyme sites

   1. Provides flexibility in inserting GOI at specific sites

3. Selectable marker gene (antibiotic resistance genes)

   1. Allows identification of successfully transformed host cells

4. Reporter gene (GFP or lacZ for blue/white screening)

   1. Allows visual or measurable confirmation of GOI expression

This is inserted to observe actual mRNA expression of GOI into mRNA and protein in target organism

Expression vector

Enumerate parts of expression vectors

• Origin of replication

• Several restriction enzyme sites

• Selectable marker gene

• Reporter genes

• Regulatory sequences

  • Drives expression → transcription/translation of GOI in host cells

  • e.g., Promoters, Shine Dalgarno sequence (RBS)

“Molecular paste” joining DNA fragments with complementary sticky or blunt ends

DNA ligase

DNA ligase creates _ bonds

• Phosphodiester bonds

• Covalent bonds between sugar and phosphate of adjacent nucleotides

Explain general process for producing recombinant DNA

1. Restriction enzyme cuts sugar-phosphate backbones in specific restriction sites, producing complementary sticky/blunt ends

2. DNA/GOI cut by the same RE from another molecule is then added

3. Base pairing occurs

4. DNA ligase seals strands

Recombinant DNA is formed by _

• Cutting plasmid vector with RE

• Cutting and isolating GOI with same RE from donor cell

• Ligating those 2 DNAs to form recombinant molecule

Stage where recombinant DNA/plasmid is inserted into host cells; replicates within host cells, producing dozens of identical copies/clones

Transformation of host cell with recombinant DNA

Enumerate and explain pros & cons of molecular cloning hosts

• Bacteria

  • Escherichia coli wmm pp

    • Pros

      • Well-developed genetics

      • Many strains available

      • Most studied bacterium

    • Cons

      • Pathogenic

      • Periplasm traps proteins G(-)

  • Bacillus subtilis enne gg

    • Pros

      • Easily transformed

      • Nonpathogenic

      • Naturally secretes proteins G(+)

      • Endospore formation simplifies culture

    • Cons

      • Genetically unstable

      • Genetics is less developed than E. coli

• Eukaryotes: Saccharomyces cerevisiae wnce pw

  • Pros

    • Well-developed genetics

    • Nonpathogenic

    • Can process eukaryotic mRNA

    • Easy to grow

  • Cons

    • Plasmids unstable

    • Will not replicate most bacterial plasmids

Enumerate and explain 6 common gene transfer methods

tbmect

1. Transformation: uptake of environmental naked DNA by competent cells

2. Biolistics: direct delivery of DNA-coated microprojectiles using high-velocity gene guns

3. Microinjection: direct delivery of DNA using fine needles

4. Electroporation: use of electrical pulses to create temporary pores in host cell membrane, allowing DNA entry

5. Conjugation (e.g., allelic exchange): transfer of DNA between cells via direct contact (pilus)

6. Transduction: transfer of DNA between cells via bacteriophage

Process where recombinant DNA within host cells are passed onto their progeny, creating population of host cells carrying cloned DNA sequence

Replication of host cells

Stage where colonies that were successfully genetically transformed are identified/determined

Screening or selection of host cells with recombinant DNA

T/F: Genetic transformation is a ‘hit or miss’ strategy

TRUE

Screening process could either be:

• Detection/isolation of the GOI via PCR, RE digestion

• Expression of selectable marker and reporter gene

  • e.g., lacZ gene for blue/white screening

  • lacZ gene encodes B-galactosidase, which leaves lactose analogs, including X-gal (chromogenic)

  • When X-gal is cleaved = blue colonies are expressed, which means

  • In case of successful transformation (insertion of GOI into vector, disrupting lacZ) = white colonies must be observed

  • Hence, failure to transform and thus insert GOI into vector, leaves lacZ functional (ultimately cleaving X-gal) = blue colonies

Corrosions accelerated by microorganisms

Microbially influenced corrosions (MICs)

2 classifications of MICs

• Metal corrosions

• Crown corrosions

• Type of MIC

• Iron, steel

• Microbes implicated sffm

  • Sulfate-reducing bacteria

  • Ferrous iron (Fe2+) -oxidizing bacteria

  • Ferric iron (Fe3+) -reducing bacteria

  • Methanogens (CO2 + H → CH4 + H2O)

Metal corrosion

Rapid form of microbial biodeterioration

Crown corrosion

Crown corrosions are often observed in _

concrete sewer tiles

A consequence of interactions between sulfate-reducing and sulfur-oxidizing bacteria

Crown corrosion

Can lead to collapse of underground wastewater transmission pipe systems

Crown corrosions

T/F: In observation of transformation, E. coli needs to be lysed to express proteins

TRUE

T/F: Proteins expressed using B. subtilis as host will be observed in cells

FALSE

Proteins expressed using B. subtilis as host will be observed in medium

T/F: All antibiotic-resistant bacteria observed are successfully transformed

FALSE

Possible that vector plasmid was empty, i.e., did not contain GOI

Explain MIC mechanism of iron corrosion by SRB (via increasing acidity/lowering pH)

• SRB produce H₂S through sulfate-reduction (SO₄^2-)

• This produced H2S then reacts with Fe2+ in the iron metal, producing FeS + H⁺

  • H₂S + Fe²⁺ → FeS + H⁺

• Increase in H⁺ leads to more acidic environment that initiates and accelerates iron corrosion

Explain MIC mechanism of iron corrosion by SRB (accelerated via bacterial consumption of H₂)

• When iron Fe⁰ oxidizes in anodic region, it forms Fe²⁺, leaving behind a corrosion pit

• Electrons produced from oxidation then migrates to cathodic region, where water dissociates into protons + hydroxide ions

• Protons are then utilized by SRB to reduce sulfate, producing H₂S, which then again reacts with Fe, producing more H⁺ that further acidifies environment, accelerating iron metal corrosion

  • H² + SO₄^2- → HS^-

What compounds indicate iron metal corrosion?

• Ferrous iron (Fe²⁺)

• Bicarbonate (HCO^3-)

• Hydrosulfide (HS^-)

Explain MIC mechanism of iron corrosion by SRB (accelerated via direct electron transfer from metal)

• Fe⁰ oxidizes into Fe²⁺, forming corrosion pits

• Fe⁰ directly donates electrons to SRB via periplasmic electron transfer complexes

• Electrons are then used by SRB to reduce sulfate SO₄^2- → HS^-

• HS^- reacts with Fe²⁺ → FeS + H, accelerating corrosion

T/F: Corrosion only happens anaerobically

FALSE

Iron-oxidizing bacteria (IOB) do it aerobically

T/F: Iron-oxiding bacteria are anaerobes

FALSE

Aerobes

Explain MIC mechanism of iron by IOB

• (A) Role of IOB in iron corrosion

  • Fe0 oxidizes into Fe2+

  • IOB catalyzes oxidation of Fe2+ → Fe3+

  • IOB utilize O2 + H2O → OH-, which increase alkalinity → accelerating iron corrosion (in oxic environments)

• (B) Interaction between SRB & IOB

  • IOB → Fe2+

  • SRB (via direct electron transfer from iron, consumption of H+ from cathodic region) reduces SO42- into HS-, which can then react with Fe2+ → FeS, accelerating corrosion

• (C) Cathodic, anodic regions

  • Fe0 oxidizes into Fe2+ in anodic regions

  • SRB reduces SO42- → HS- in cathodic regions

  • Both combine to create localized corrosion pits

Explain MIC mechanism of iron corrosion by IRB

• IRB reduces Fe³⁺ → Fe²⁺ under anaerobic conditions

• IRB reducing Fe³⁺ → Fe²⁺ ,which can then react with H2S → FeS + H+, further accelerating corrosion

T/F: IRB are anaerobes, IOB are aerobes

TRUE

Explain MIC mechanism of iron corrosion by methanogens

• Electrons released from iron oxidation is used by methanogens to reduce CO2 → CH4

• Fe0 oxidation → Fe2+, which can then react with H2S → FeS + H+, further accelerating corrosion

Explain crown corrosion

• In anoxic region (submerged in water), SRB reduces SO₄^2- → H₂S, which is then released up towards crown

• In oxic region, SOB then oxidizes H₂S, forming sulfuric acid

  • H₂S + O2 → H₂SO₄

• Sulfuric acid → lowers pH, further accelerating corrosion

T/F: SOB alone can cause crown corrosion

FALSE

SRB produces H2S through sulfate reduction. H2S is then oxidized by SOB, producing H2SO4 that acidifes environment

T/F: SRB is aerobic, SOB is anaerobic

FALSE

SRB is anaerobic, SOB is aerobic