UNIT 3 AOS1

what are plasmids?

a small, circular piece of double-stranded DNA that is able to reproduce independently and may be taken u by cells (usually bacteria).

can also carry advantageous genes such as antibiotic resistance

what is an origin or replication (ORI)?

A DNA base sequence in a plasmid in which DNA replication begins

what are recombinant plasmids?

plasmids that carry foreign DNA

what are the features found in a recombinant plasmid?

• ORI

• antibiotic resistance gene

• multiple closing site

• promoter regions

• selectable marker

• screening marker

what are vectors?

an agent (plasmids) used to transfer pathogens or genes between cells and organisms

what is the function of screening markers?

allows fro the confirmation that a plasmid is actually recombinant

what is the function of a selectable marker?

a gene introduced that allows for identification and selection of certain cells

how are recombinant plasmids formed ?

1. The DNA of the plasmid is endonucleased in order to create sticky ends. This changes the plasmid from circular to linear.

2. The foreign DNA fragments are prepared using the same endonuclease so that the foreign DNA has sticky ends complementary to the cut plasmid. Reverse-transcriptase can be used to remove introns.

3. The foreign DNA fragments and the plasmids are mixed, and, in some cases, their ‘sticky ends’ pair by using weak hydrogen bonds. A recombinant plasmid has been created.
   (Other pairings will also occur, such as cut plasmids resealing themselves so that they are not recombinant plasmids).

4. The joining enzyme, DNA ligase, is added to permanently bind through covalent phosphodiester bonds.

what is bacterial transformation?

the process in which bacterial cells take up segments of foreign DNA that become part f their genetic make-up

• low success rate

how can success rate be increased when transferring recombinant plasmids into bacterial cells?

• electroporation

a technique that used brief exposure to host cells to an electric field to enable the entry of segments of foreign DNA into the cells

• heat shock

a technique where cells are suspended in an ice cold solution and then moved into a warm solution to increase plasma membrane fluidity

what is foreign cDNA?

the DNA form the gene of interest that we want expressed in bacteria

why do we use bacterial transformation?

to express and extract proteins that are useful to humans in a cheaper, safer and more ethical method

what is the process of isolating recombinant DNA?

1. Bacteria are grown up agar plates containing the anti-biotic, to which they normally have no resistance

2. Those that survive and grow have taken up the resistance gene and the gene of interest

3. These transformed bacteria can be highlighted by adding a fluorescent substrate

4. These bacteria are then selected and cultivated and the expression of the foreign DNA will then provide the new protein

5. The protein produced is collected, purified and concentrated ready for use.

what is the process of the production of human insulin?

1. Preparation: The human insulin gene and a bacterial plasmid (circular DNA) are cut using the same restriction enzyme to create complementary sticky ends.

2. Ligation: The insulin gene and plasmid are mixed with DNA ligase, which joins them through covalent phosphodiester bonds to create a recombinant plasmid

3. Transformation: The recombinant plasmid is added to a culture of bacteria. Using heat shock or electroporation, the bacteria are encouraged to take up the plasmid from their environment.

4. Selection: Not all bacteria will take up the plasmid. Scientists use antibiotic resistance genes (already on the plasmid) to kill off any bacteria that didn't successfully transform - fluorescent substrate can also be used here for identification.

5. Production: The transformed bacteria are grown in large vats. Because the bacteria now contain the human gene, they start expressing the protein and producing human insulin.

PLTSP

what is insulin?

insulin is a peptide that consists of two polypeptide chains, alpha and beta sub units within the pancreas that release insulin

what is genetic engineering?

changing an organisms genome using recombinant technology

what is a genetically modified organism?

any organism that has been changed by genetic engineering

what are transgenic organisms?

organisms that carry in their genomes one or more genes artificially introduced from another species

what does genetically modifying genes do and what outcome does it have on the organism/s?

• it can silence. remove , add or alter the gene

→ outcomes:

• this can be used to treat genetic diseases, changing desirable traits in animal and plants, increasing genetic diversity yields introducing new alleles

• can improve crop productivity

• can resist disease

what is integrity for GMO?

manufactures might need to clearly label their products as GMOS, allowing consumers to be better informed

what is justice for GMO?

GMOS might create inequity between larger agricultural companies who have the resources to genetically alter their crops vs a small family business

what is beneficence for gmos?

there are positive health outcomes for people who consume GM foods that have been nutritionally enhanced

what is non-maleficence in GMOS?

GMOs might cause unintended disruptions to the food web, such as insect resistant crops that may alter population levels of different pests so they must be tested heavily before being released to ensure as minimal of a disruption to these factors

what is respect in GMOS?

it is important to promote the right individuals have to freely choose whether or not they use GMOS, and be provided wiht equal representation of alternatives.

what are consequence based ethics?

aims to maximise postive outcomes and minimise negative outcomes

what are duty/rule based ethics ?

encourages people to follow rules which society had agreed upon

what are virtue based ethics?

looks at the character of a person performing the actions and what constitutes as good behaviour

what are the good about GMO?

some of the argument FOR the use of GMO include:

• herbicide or pesticide resistance

• superfoods such as golden rice

• health

• increased yield

what are the bad about GMO?

some of the arguments against the use of GMO include:

• agribusiness monopolies ( one company owns the rights to produce the GM seed)

• reduction in genetic diversity

• cross polination

• general religious/ethical concerns

what is DNA finger printing?

a technique for identifying DNA from different individuals based on variable numbers of short tandem repeats (str) in DNA segments near the ends of chromosomes

what is gel electrophoresis?

a biotechnology for sorting a mixture od DNA fragments through an electrical field on the basis of fragment length

what is DNA sequencing?

identification of the sequence of bases along a DNA strand

what is the process of separating fragments by gel electrophoresis?

1. The DNA sample with fragments of varying sizes is combined with DNA loading dye.

2. This mixture is placed in a well at one end of a slab of jelly-like supporting material, known as an agarose gel.

3. This agarose gel is immersed in a buffer solution (salt solution).

4. The gel is then exposed to an electric field with the positive (+) pole (anode) at the far end and the negative (−) pole (cathode) at the starting origin. The DNA is attracted to the positive pole.

5. Smaller fragments move through the agarose gel faster compared to larger fragments.

6. These fragments appear as bands on a gel which can be interpreted in various ways. This usually needs to be observed under UV light.

what are some factors that affect migration of DNA?

• DNA is NEGATIVELY charged, therefore it travels to the positive cathode

• the size of each piece of DNA gives information about the number of bases present

• high voltage → DNA moves faster through the gel

• length of time → the longer voltage is applied for, the further the distance the DNA with travel

• gel concentration - denser agarose gel (small pores in gel), the slower the DNA fragments will move

what is a probe? + an example

a single-stranded segment of DNA or RNA which carries a radioactive/ fluorescent label wiht a base sequence complementary to that in the target DNA

• ethidium bromine

what would happen if loading dye wasn’t added during electrophoresis?

the bands/dna sample would sink down the wells and you wouldnt be able to see the samples

why might an individual have a thicker band?

the individual might have the exact same amount of strs as both parents or the specific section have a higher concentration of the dna sample/str in that section

what is the purpose of running an allele ladder as part of the gel?

Is an allele ladder required in all situations?

As we have different STRs in samples of DNA the allele ladder X as a stopper during electrophoresis. The ladder contains all common alleles and where the sample stops refers to how many repeats there are.

• not required in all situations - mainly in forensics and paternity tests

what are short tandem repeats (STR)?

sections of DNA that are comprised of short, repeating bases that are unique to each individual

• used in crime scene or in paterinty tests

• highly polymorphic

What are ribosomes?

Sites of translation in which amino acids are joined to form polypeptides

Within a cell ribosomes are attached to membrane channels known as the endoplasmic reticulum or in free cytosol

What is the rough endoplasmic reticulum?

Considered rough when ribosomes are attached to his channels and highlights an interconnected system

• folding and modifying proteins before transporting them to various sites throughout the cell

What is the Golgi apparatus?

An organelle which packages and tags proteins before sending it to secretory vesicles

What are vesicles?

Organelles which break free from the protein cargo from the Golgi apparatus which moves to the plasma membrane of the cell infuses to discharge the protein

What is the process of the protein secretory pathway?

Nucleus - ribosome - roughER - transport vesicle - Golgi apparatus - secretory vesicle

What is the coding region?

A part of the gene that contains the coded information for making a polypeptide chain

What are the flanking regions?

Regions located either downstream or upstream of a region of a gene

What are promoter regions?

Regions located at the beginning of a gene which are rich in A,T nitrogenous bases and CAAT which makes transcription process more efficient

What occurs at the promoter region?

Where transcription factors and RNA polymerase bind to initiate transcription

What is a leader region?

Contains sections known as attenuators that are involved in gene regulation

What are operator regions?

In between the promoter and the gene being transcribed is a binding site for repressor proteins (proteins which prevent transcription) to continue the process of transcription only present in prokaryotic cells

What is the differences between eukaryotic and prokaryotic gene structures?

In prokaryotic genes there are operator regions however in eukaryotic gene structures there are no operator regions and instead have enhancer regions which can turn on or off the expression of a gene

What is a repressor?

Proteins which prevent transcription

What is a Tata box?

A sequence of the promoter region, which is where the RNA polymerase binds

What is the CAAT box ?

A region which makes the process of transcription more efficient

What are UTR‘s (untranslated regions) ?

The UTR’s tell the guanine cap and the poly a tail where to bind

What is the general sequence of regions in a eukaryotic structural gene?

enhancer + silencer —— promoter region with the tata box, the 5’ UTR, the start codon, exons and introns, stop codon, 3’ UTR —— terminator

What is the general sequence of regions in a prokaryotic structural gene?

Promoter region, operator region, 5’ UTR (leader), start codon, coding sequence, stop codon, 3’ UTR(trailer), terminator.

What are enhancers and silencers?

short, specialized DNA sequences acting as molecular switches to control gene expression by increasing (enhancers) or decreasing (silencers) transcription rates

What is a genetic code?

The instructions used by cells to translate encoded inf into proteins

What are cells?

Cells are the basic structural and functional unit of life which vary in size.

What are the types of eukaryotic cells?

• Animalia

• Plantae

• Fungi

• Chromista

• Protozoa

What are the types of prokaryotic cells?

• Bacteria

• Archea

What is similar between prokaryotic and eukaryotic cells?

• Both have DNA in their genetic material

• Both produce protein through the same mechanism

What is a nucleus?

A membrane surrounded organelle which houses chromosomes and transcribes genetic information

What are ribosomes?

Consists of two components smaller and larger ribosome slap units which are involved in protein synthesis

What is the smooth endoplasmic reticulum?

Synthesis of lipids metabolism of carbohydrates and calcium storage

What is the rough endoplasmic reticulum?

Synthesis of specific proteins from bound ribosomes

What is the Golgi apparatus?

Modify sorts, tags and distribute proteins to be secreted via vesicles

What is the plasma/cell membrane?

Consists of the phospholipid bilayer controlling the entry and exit of raw materials and waste

What are lysosomes?

Membrane enclosed cells which breakdown cellular waste

What are vacuoles?

Large membrane bound organelles found only in plant cells which digest, store, dispose waste and control water balance

What are peroxisomes?

An enzyme which transfers hydrogen to water producing hydrogen peroxide

What is the mitochondria?

A double membraned site of cellular respiration and ATP synthesis

What is a chloroplast?

A double membrane organelle which conducts photosynthesis in plants only

What are the types of nucleic acids?

DNA and RNA

What is DNA?

Deoxyribose nucleic acid is a polymer located in chromosomes in the nucleus with the subunits of deoxyribose sugar phosphate group and nitrogenous bases.

What is RNA?

Rio nucleic acid is formed against the DNA template strand and is a single chain with rubbers, sugar and thymine changed to uracil

What are the types of RNA?

• Messenger RNA mRNA

• Ribosomal RNA rRNA

• Transfer RNA tRNA

What is Messenger RNA?

Carries a copy of genetic information from within the nucleus into the cytoplasm to the ribosomes for the message to be translated into a particular protein

What is ribosomal RNA?

A stable form of RNA within ribosomes

What is transfer RNA?

A form of RNA that attaches to amino acids with its anticodon and carries them to ribosomes during translation.

How are nitrogenous spaces held together?

Adenine - Thymine is held with 2 hydrogen bonds

Guanine - Cytosine is held with 3 hydrogen bonds

How can you identify which end of a DNA’s backbone is the five end or the three end?

If the sequence ends with a phosphate group, then it must be the five’ end and if it ends with the OH group, it must be the three’ end.

What does it mean for DNA to be anti-parallel?

One side of the double helix strand goes in a 5 to 3 direction whereas the other strand goes in a 3 to 5 direction allowing the nitrogenous spaces to bond

What is RNA polymerase?

An enzyme which transcribes the mRNA sequence into RNA in a 5’ to 3’ direction

what is DNA polymerase?

an enzyme essential for DNA replication in a 5’ 3’ direction

what are endonuclease’s?

restriction enzymes that cut at specific sites within DNA molecules in a controlled method.

what are blunt ends?

ends of a DNA fragment wiht no overhanging bases after being cut directly opposite of each other

what are sticky ends?

ends of a DNA fragment with overhanging bases after being cut not opposite each other - complementary

How many cuts and fragments are produced if the DNA is linear?

if the DNA was cut twice there would be three fragments produced

How many cuts and fragments are produced if the DNA is circular?

if the DNA was cut twice there would be two fragments produced

what is DNA ligase?

an enzyme which catalyses the joining of pieces of double stranded DNA at their sugar phosphate backbone.

How does DNA ligase ‘glue’ the DNA sugar phosphate backbone?

forms a phosphodiester bond between the two fragments of DNA, joined by the 3’ and 5’ end

what are polymerases?

enzymes that allow for the synthesis of nucleic acids

what are recognition sites?

short sequences of nucleotides which are bound with restriction enzymes

what is Taq polymerase?

a type of DNA polymerase which is isolated from a heat-resistant bacteria used to add complementary nucleotides to single stranded DNA in a 5’ to 3’ direction and is essential for amplifying DNA

how do you obtain endonuclease’s?

endonuclease’s are naturally found in bacteria, where they help cut out any DNA inserted by the bacteriophage.

What is the structure of amino acids?

Polypeptide’s are large molecules built of subunits called amino acids. The general formula for an amino acid contains the amino group the variable group and the carboxyl group

What are the monomer of proteins?

Amino acids are the monomer of proteins with 20 different amino acids each with a different variable group

How does the formation of a polypeptide chain occur?

Proteins assembled from amino acids that are joint together by peptide bonds