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Biotechnology

is the development of technology which utilizes biological systems, living organisms, or components of living organisms to create different products

Genetic engineering

is the use of in vitro techniques to alter genes in the laboratory. These altered genes can be reintroduced into the organism, and the effects can be determined.

Heterologous expression

is the insertion of a gene into an organism that does not normally contain the gene, to give this organism new functionality

Polymerase chain reaction (PCR)

is biotechnological technique that allows you to amplify a specific region of DNA in vitro (in a test tube).

thermocycler

changes the temperature of the reaction mixture to a set number for a set amount of time. PCR)

PCR

_________ mimics DNA replication and shares similar mechanism. It also shares some, but not all, of the enzymes

Template DNA

DNA which contains the target gene. Acts as the template strand for the reaction

Primers

Single-stranded DNA (ssDNA) which bind complementary regions either side of the target gene. Provide a 3’ OH to enable DNA synthesis. Replaces primase

DNA polymerase

The enzyme which performs the DNA synthesis reaction, like that used in replication

dNTPs

Deoxyribonucleotide triphosphates which serve as the building blocks of DNA and provide energy.

Cofactors

Any of the necessary factors required for DNA replication (i.e. Magnesium).

Polymerase chain reaction - Components

Template DNA, Primers, DNA polymerase, dNTPs, Cofactors

Denaturation

1. The hydrogen bonds between the DNA base pairs are broken, resulting in single-stranded DNA (ssDNA) molecules.

Annealing

2. The primers anneal to the target sites on the DNA.

Extension

3. DNA polymerase uses the primers as a starting point to amplify DNA.

two

PCR reactions always require _____ primers. These are referred to as forward and reverse primers.

Reverse Primer

The primer which binds at the 5’ end upstream of the “coding” strand (strand containing genes of interest).

Forward Primer

The primer which binds at the 5’ end upstream of the “template” strand (strand which does not contain gene of interest).

DNA polymerase

The _______ used in the PCR is from a thermophile, which means it is stable at high temperatures (heat-stable). This prevents denaturation of the protein during the PCR.

isolation

PCR's ability to amplify DNA enables _______ of DNA from sources that would otherwise be impossible: • Mummified human remains. • Fossilized Plants • Fossilized Animals • Single microbial cells.

Gel Electrophoresis

Method of separating molecules based on their size-to-charge ratio using an agarose gel

Agarose

Pure seaweed extract which forms a gelatinous gel, acts as a molecular sieve, slowing down larger particles.

Buffer

Carries the electrical current required for separation.

negative, positive

DNA contains a uniform ______ charge due to the presence of the phosphate backbone. DNA will move towards the ______ charge.

Ethidium Bromide (EtBr)

contains a structure that resembles a base-pair. It inserts into the DNA between base pairs, when inserts into the DNA causing it to fluoresce under UV light.

longer

The ________ the DNA the slower it moves in agarose matrix, meaning it will appear closer to the wells of the gel.

brighter

The more DNA present the more EtBr is present, this will result in a ______ band.

genome

The _______ is all the genetic information that defines an organism

genome

Various processes are encoded by the _____: • Energy and nutrient sources (catabolism). • Growth products (biosynthesis). • Factors that promote fitness (ecology). • Molecular adaptations (evolution)

Open Reading Frame (ORF)

A region of DNA that begins with a start codon and ends with a stop codon.

Complexity

________ often, but not always, increases with increasing genome size.

smallest

Bacteria generally have the _______ genomes of any living organism, while plants have the largest.

genome

The ______ of an organism can be derived using the following steps:

1. Sequence: Determining the order of the bases within DNA. 2. Assemble: Piece together different parts of the genome in the correct order.

3. Annotate: Convert this raw data into a list of genes whose function may or may not be known.

Bioinformatics

is the process of using a computer to store and analyze the sequences and structures of DNA and proteins.

Sanger sequencing

this technique uses DNA synthesis to determine the sequence of a region of DNA.

Dideoxyribonucleotide triphosphates (ddNTPs)

They lack a 3’ OH. This means there is no way to extend the chain past, further monomers cannot be added. They are referred to as terminal nucleotides. (sanger)

ddNTPs

Integration of _____ is random, with millions of DNA strands made during sequencing it is inevitable that a _____ will be present at every single residue on different strands. (sanger)

smaller

The _____ the fragment the earlier it comes in the sequence. The template strand is complementary to the sequence obtained as synthesis is 5’ – 3’.

primer

Sanger sequencing requires the use of a _____. This must bind a known sequence

Limitations of Sanger sequencing

Limited to around 800 nucleotides per reaction.

• Can only sequence from known sequences.

• Cannot be used in large-scale genome sequencing.

annotation

The _____ relies on finding open reading frames (ORFs), which begin at a start codon (ATG) Sanger

ribosome binding site (RBS).

Genome annotation: Once the start codon is found the software will look for a potential protein sequence ending at a stop codon.The software will then determine whether there is a canonical ___________ Sanger

ORF

ribosome binding site (RBS), a start codo, protein sequence ending at a stop codon Sanger

ORF

Genome Annotation: If an ____ is determined to be valid the DNA sequence is translated into a protein sequence Sanger

redundancy

codons have ________, several codons encode the same amino acid

horizontally transferred

This difference in codon frequency usage can indicate which genes have been __________ ________

Nucleases

Enzymes that digest (break apart) nucleic acids via hydrolysis.

Exonuclease

Cleaves from the ends of the molecule.

Endonuclease

Cleaves in the middle of the molecule.

Restriction endonuclease

Enzymes that hydrolyze double stranded DNA at specific spots on opposite strands.

palindromes

This means they are identical when read 5’ → 3’ on both strands

Restriction endonucleases

_____ Can result in the formation of sticky or blunt ends

Sticky end

Short, single-stranded stretches at the

end of the double-stranded DNA, the bases of the ssDNA want to hydrogen bond to complementary bases

sticky ends

Complementary _______ can hydrogen bond to one another. This joins two separate pieces of DNA into one large piece of DNA.

Recombinant DNA

DNA that is formed by joining two different pieces of DNA together. Can be constructed utilizing sticky ends.

restriction

You can digest two pieces of DNA with the same _________ enzymes and then mix them.

ligase

Nicks in the backbone are sealed with DNA _______

Example: You can join a PCR product with a plasmid.

Plasmids (vectors)

Small, circular, self-replicating DNA molecules that contain genes for antibiotic resistance and are used in cloning.

Selection

Process of isolating bacteria which contain a genetic marker of interest.

Antibiotic resistance

The ability to grow in the presence of a lethal antibiotic. The most common selection marker. Kanamycin). (Ampicillin, Tetracycline,

resistance marker

By growing cells in the presence of a selectable marker only cells containing the ___________ (on the plasmid) will survive.

isolated

Plasmids can be ______ from bacteria: • Grow bacteria containing the plasmid in the presence of selectable marker. • Lyse the cells. • Purify the plasmids using a molecular biology kit.

transformation

Plasmids can be taken up by cells that have been made artificially competent via ________

Multiple cloning site (MCS)

Region of a bacterial plasmid with a sequence that can be cut by multiple restriction enzymes

Inducible promoter

A promoter which triggers gene expression under certain, inducing, conditions.

Outcomes of recombination

The plasmid can successfully integrate the DNA of interest. • The plasmid can re-bind to itself and become recyclized. This means your recombination was unsuccessful as it does not contain an insert.

pUC 19

_____ plasmid contains a gene for ampicillin resistance and the gene encoding ß-galactosidase (LacZ).

Ampicillin resistance

Enables growth in the presence of ampicillin (selection marker).

β-galactosidase

Breaks down lactose if expressed, it can break down the X-gal. When the colorless X-gal is broken down it creates a blue dye.

multiple cloning site of pUC

is inside the lacZ gene. This means that if the DNA is inserted it disrupt the reading frame of lacZ. This makes β-galactosidase non functional

recombination was successful

: Insert present → lacZ disrupted → White colonie

recombination was unsuccessful

Insert absent → lacZ intact → Blue colonies

Allele

A different version of a gene. I.e. Blue vs Brown Eyes.

Evolution

involves selection for or against mutations based on how it affects the organism's fitness

Fitness

is the reproductive success of an organism

Mutations

are usually deleterious (bad) or lethal. However, in some cases they can be neutral or beneficial

Deleterious mutations

are selected against and disappear from a population

Neutral mutation

have no selective pressure and will change their frequency randomly (genetic drift).

Beneficial mutations

are selected for and will increase in abundance in a population.

Gene duplications

the second copy of the gene can be mutated without immediately impacting the cell’s original metabolic processes, as one functional gene still exists.

genome

gene duplication may gain a new functionality and become beneficial to the cell. The cell will now select for this gene as well and it will be retained in the_____

Homolog

set of genes which are descended from a shared common ancestor

Orthologs

Genes that are derived from a common ancestor and therefore have a similar sequence and function

Paralogs

Genes which are homologous but do not share the same function

orthologs

An enzyme which metabolizes glucose (sugar) in E. coli and one that metabolizes glucose (sugar) in S. aureus are ______.

paralogs

An enzyme which metabolizes glucose (sugar) in E. coli and one that metabolizes galactose (sugar) in E. coli are ________.

evolution of interdependency

Gene deletion can involve the loss of a gene encoding a product. If this product is essential, this is lethal, however in microbial populations other organisms in the population may compensate for the loss of production.

horizontal gene transfer

can result in addition of genetic material into a genome from an unrelated microbial species.

complicate

Horizontal gene transfer can ______ phylogenetic analysis into microbial evolution, as there is transfer from distant branches of the tree of life.This makes some species appear more closely related than they are.

Comparative genomics

Comparison of the differences in the genomes of two organisms

strains

Members of a single species that have large genomic differences

Phylogeny

Evolutionary history of organisms.

Lineage

is a continuous line of descent from a common ancestor

Root

is the closest ancestor to all organisms in a phylogenetic tree; it is generally theoretical.

Node

represents an ancestor which diverged into two different lineage

Branches

represent the number of evolutionary changes that have occurred during evolution

Building a phylogenetic tree

1. Align the DNA sequences of a shared gene in target organisms.

2. Compare these target genes to one another, noting differences.

3. Organize the target genes based on their similarity.

rRNA of the small subunit (SSU)

genes encoding the ______ are ideal candidates for construction of the tree of life

Highly conserved regions

provide a stable reference point to align sequences from all organisms. tree of lif

Variable regions

enables distinction between closely related organisms. tree of life