[BIO 120.11] Module 4 Part 2

Genetic Engineering

Uses laboratory-based technologies to alter the DNA makeup of an organism

Recombinant (rDNA) DNA technology

What is the technology often used to perform genetic engineering?

TRUE

T/F: Aside from rDNA technology, there are other ways to do genetic engineering.

FALSE

T/F: Genetic engineering is basically just rDNA technology

TRUE

T/F: Although microorganisms are primary, other organisms may be used as tools in development of genetic engineering.

FALSE

T/F: Genetic engineering involves the usage only of microorganisms as they are the only microorganisms whose genomes can be readily altered.

Recombinant, GMO, Transgenic

What are 3 other terms for genetically engineered organisms?

Recombinant DNA

Genetic material that contains the foreign gene

Recombinant DNA

Gene of interest and vector

Recombinant Organism

The host that carries the recombinant DNA molecule

TRUE

T/F: Any organism can be a source of gene for cloning as long as you know the sequence of your target gene.

FALSE

T/F: The source of your target gene must be the same species as the host recombinant organism.

Isolation of gene to be cloned
Ligation of DNA into a vector
Transformation of a host cell with the recombinant DNA
Selection or screening of a host cell with the recombinant DNA

What are the 4 Steps for Gene Cloning?

Restriction Enzymes
PCR
Artificial Gene Synthesis

What are 3 ways to isolate a gene to be cloned?

Restriction Enzymes

Molecular scissors that accurately and reproducibly cut genomic DNA into fragments

FALSE

T/F: Restriction enzymes are exonucleases that cut within DNA

TRUE

T/F: Restriction enzymes are endonucleases that cut within DNA

FALSE

T/F: Restriction enzymes are endonucleases that cut within the plasmid

Gene should be near or between restriction sites

In order for the target gene to be isolated via restriction enzymes, what must be the primary consideration?

HindIII

Restriction Enzyme that cuts between 2 adenines in an AAGCTT sequence

Alu1

Example of a restriction enzyme that produces blunt ends

BamHI

Restriction Enzyme that cuts between 2 guanines in a GGATCC sequence

Sau3AI

Restriction Enzyme that cuts beside a G nucleotide in a GATC sequence

AluI

Restriction Enzyme that cuts in the middle of an AGCT sequence

Isolating, Copying

PCR is a rapid method for doing what things to a gene?

TRUE

T/F: PCR can amplify specific DNA sequences that are present in small quantities even if mixed with many other DNA molecules

FALSE

T/F: PCR can amplify specific DNA sequences provided that it is not mixed with other DNA molecules

Denature DNA (92 - 95 deg C)
Anneal Primers (45 - 65 deg C)
Extend Primers (65 - 75 deg C)

What are the 3 steps in a PCR reaction

Hydrogen bonds

What kind of bonds are destroyed in the denaturation step of PCR?

Gene synthesis by polymerase cycling assembly (PCA)
Gene synthesis by ligation

What are 2 ways in which artificial gene synthesis can be done to isolate a target gene?

Phosphodiester bond

Bond that is synthesized by ligase

Phosphodiester bond

Covalent bond that links a 5’ phosphate with the upstream 3’OH group, holding the DNA backbone together

Hydrogen bond

Intermolecular bond that holds nitrogenous bases together

Hydrogen bond

Bond that must be broken prior to DNA replication and transcription

Ligation of DNA into a vector

Step in gene cloning wherein a gene of interest is joined to a vector

Plasmids
Bacteriophage
Cosmids
YACs
BACs

What are 5 types of vectors?

Cosmid

Combination of a plasmid and a phage

YACs, BACs

What kind of vectors must be used if we want to work with larger genes?

Cloning Vectors

Carrier of specific gene of interest into the host cell

Cloning Vectors

Make numerous copies of the gene

Origin of replication
Several restriction enzyme cleavage sites
Selectable marker gene
Reporter gene

Composition of Cloning Vectors

Antibiotic Resistance Gene

What is an example of a selectable marker gene?

lacZ gene

What is an example of a reporter gene?

Origin of replication
Several restriction enzyme cleavage sites
Selectable marker gene
Reporter gene

Composition of Expression Vectors

Expression Vectors

Vectors needed in actual expression of the gene into mRNA and protein in the target organism

Regulatory sequences

What is a component of expression vector that cannot be found in cloning vectors

Shine Dalgarno sequence

Ribosome binding site for bacteria

DNA ligase

Joins DNA fragments that have complementary sticky ends or blunt ends

Covalent bonds between sugar and phosphate of adjacent nucleotides

DNA ligase creates what kind of bonds?

Clones

What is produced when the DNA is transformed into a host cell and that cell produces identical copies?

E. coli, B. subtilis

What are 2 common hosts for molecular cloning?

Well developed genetics
Many strains available
Most studied bacterium

3 advantages of using E. coli as a host cell

Potentially pathogenic
Periplasm traps proteins

What are 2 disadvantages of E. coli as a host cell?

Easily transformed
Nonpathogenic
Naturally secretes proteins
Endospore formation simplifies culture

What are 4 advantages of using B. subtilis as a host cell?

Transformation
Biolistics (projectile gun)
Microinjection
Electroporation (electrocute cells to create pores)
Conjugation (ex. allelic exchange)
Transduction (via phage)

What are 6 examples of gene transfer methods?

TRUE

T/F: The recombinant DNA molecules within a host cell is passed on to all of their progeny

FALSE

T/F: The recombinant DNA molecules within a host cell is only selectively passed on to all their progeny (hit or miss)

FALSE

T/F: Replication of host cell with recombinant DNA is a hit or miss strategy

TRUE

T/F: Genetic transformation is a hit or miss strategy

FALSE

T/F: Increasing the precision of the method allows genetic transformation to be more ensured

Detection or isolation of the rDNA – PCR, RE digestion
Expression of the selectable marker and reporter gene

What are 2 ways in which screening process for genetically transformed colonies can be done?

TRUE

T/F: In a screening process, the colonies that are antibiotic resistant are not yet automatically positive for the recombinant DNA

FALSE

T/F: In a screening process, the colonies that are antibiotic resistant are positive for the recombinant DNA

Microbially Influenced Corrosion

Corrosion accelerated by microorganisms

Metal Corrosion (Iron, Steel)
Crown Corrosion (Cement)

What are 2 types of Microbially Influenced Corrosion (MICs)?

[SRB] Sulfate Reducing Bacteria
[IOB] Ferrous (Fe2+) Iron Oxidizing Bacteria
[IRB] Ferric iron (Fe3+) Reducing Bacteria
Methanogens

What are 4 microbes implicated in metal corrosion?

Bacterial consumption of H2 accelerates oxidation of metallic iron
Direct electron transfer from metal

In MIC Mechanism of Iron by SRB, what are the 2 ways?

FeS, 3 FeCO3

What are 2 corrosion products in MIC Mechanism of Iron by SRB via direct electron transfer?

SO(4, 2-) to HS-; sulfate to sulfide

With electrons, what does SRB do to sulfate?

Fe2+ + H2S

Reaction between these 2 compounds result to corrosion [SRB]

Iron Oxidizing Bacteria

What bacteria usually works with SRB to do MIC?

Oxidation of Fe2+ to Fe3+

What reaction is done by IOB?

Reduction of Fe3+ to Fe2+

What reaction is done by IRB?

SRB and IRB are anaerobic, IOB is aerobic

Among the 3 bacteria implicated in MIC, which among them is anaerobic and aerobic?

The electrons released in iron oxidation (Fe0 to Fe2+) are used by methanogens for CO₂ reduction to CH4. The oxidation of iron leads to its degradation.

What is the basic principle of MIC by methanogens?

Biodeterioration

Crown corrosion is a rapid form of?

Concrete sewer tiles

In what equipment is crown corrosion common?

SRB and SOB

What bacteria are implicated in crown corrosion?