GtP

What is a recombinant protein and why is it made?

A recombinant protein is a protein made via genetic engineering, used in diagnosis, research and therapeutic

What is a heterologous system?

This is where a protein is produced, in an organism that would not typically produce that protein. Typically referred to as a bioreactor

pETBlue-1 is under the control of which set of genes? (Found in bacteria)

Under the regulation of the genes which regulate lac operon, found in E. Coli

What does the blue area signifying?

It is showing the t7 promoter, which regulates the transcription in the bacteria, which is used for the expression of recombinant proteins

What is the yellow area signifying?

This is the f1 origin, where transcription, replication and repair, using ssDNA. Location where bacteriophage was inserted into the bacteria

The green area is signifying what?

It is representing he LacZ gene, which can be altered to produce different proteins, compared to beta-galactosidase

What is the grey area signifying?

It is the origin of replication, where proliferation in the cell first begins

The orange area is signifying what?

It is showing which different types of foreign DNA can be entered, to alter the recombinant protein produced

What is the red area signifying?

It shows where the plasmid will begin to undergo bacterial resistance

What is a replicon?

Allows the bacteria to replicate the plasmid

What can occur from high plasmid numbers in a bacterium?

A metabolic burden, as it will cause a decrease in the bacterial growth rate

What is the lac operon repressor protein?

It is binds to the lactose operator site (LacO) and inhibits the production of beta-galactosidase from the lacZ gene

How is beta-galactosidase produced?

When lactose is present, a small amount is converted to allolactose, which binds to the lac repressor protein, preventing the inhibition of the Lac operator site (LacO), and causing β-Gal to be produced.

If E. Coli has been genetically modified so that viral T7 RNA polymerase is under control of the Lac Operon, what’re the steps that would be required to produce a recombinant protein?

After feeding the E. Coli lactose and IPTG, Lacl is bound to, unable to do anything, as lactose binds to the Lac operator site (LacO) activating the expression of RNA polymerase. RNA polymerase binds to the T7 promoter site in the plasmid, causing it to be translated, and proteins are expressed.

What is IPTG and it’s function?

Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a chemical mimic of allolactose, however, it cannot be broken down due to the presence of a sulphur atom, making it immune to hydrolysation

White colonies shows what?

It shows that plasmid has been successfully been integrated with the foreign DNA in component cells due to a lack of beta-galactosidase to break down the X-gal.

What is ecoRI, ecoRV and pstl, and their role on bacterial DNA? Also what are the bacteria’s own ways of cleaving it’s own DNA?

These are restriction enzymes, cutting specific parts of the DNA, typically working via genetic engineering. The bacteria’s own way to cut it’s own DNA is via endonucleases.

When restriction enzymes are methylated, what does this cause and why does it happen?

This causes restriction enzymes unable to damage the bacteria’s own DNA during cleavage. This happens due to, bacteria attempting to cut foreign DNA, but does not want to damage it’s own.

What does the arrow indicate?

Where cleavage will occur

Role of DNA ligase, and when it is used for steps to creating recombinant proteins?

It reattaches phosphodiester bond between nucleotides, where cleavage has occurred, used in the final step for construction of recombinant proteins

Which is better when trying to gain genomic information on a protein inside the human body?
Genomic DNA library or cDNA library? And why?

cDNA, as it just shows mRNA which would’ve made up the protein, whilst the human body is made up of 98% of non-coding DNA

Proteins which aid effectiveness of the binding of RNA and initiation of transcription would result in what?

A greater increase in gene expression

How does Lac Operon affect gene expression?

Low levels of glucose also releases cAMP, binding to CAP protein, which then binds to CAP operator DNA to increase transcription of lac operon, IF lactose is present too

What are some proteins which plasmids can be engineered to produce? (Done in a previous lab, 2nd year, 1st semester)

Human factor IX, absent in those with haemophilia B

When using T7 (a bacteriophage) what is required from the E. Coli for gene expression?

They need to express bacteriophage RNA polymerase, as the T7 promoter is required for the desired recombinant protein

Different types of gene expressions in complex eukaryotes

Constitutive expression: Genes are expressed all the time, in every cell, and are vital for everyday life
Temporal expression: These genes are expressed in the organism, at certain times
Spatial expression: Genes are expressed in specific areas
Responsive expression: Genes are expressed by external or internal stimuli

Haemophilia, which only affects males, would follow which hereditary pattern

X-linked recessive

What is cDNA?

It is called complementary DNA, and is copied / converted from mRNA

Ways to synthesis more cDNA?

Random primers can be used, which are an alternative to oligo-dT primers, but concentration levels can be controlled based on the level of expression wanted. These primers can bind to multiple points along the RNA sequence. Random hexamers (6 nucleotides) and nonamers (9 nucleotides) can also be used. Reverse transcriptase is then used to change the RNA to cDNA.

What is real time PCR, it’s role and 2 examples?

It uses the PCR method alongside the use of fluorescence to monitor products made during each cycle of the PCR test like nucleic acid sequences. 2 main examples are SYBR green and Taqman probes.

Difference between standard PCR test and real time PCR test?

Real time measures each cycle, while standard only measures at the end.

What is a microarray, and what is used to detect the gene’s present?

A solid substrate that contains collections of ssDNA, a with one dot containing a picomole of DNA. In most examples, all expression genes would be used. Fluorescence is also used, which binds to the sample, with a greater signal representing a greater amount of target present.

Use of microarrays?

RNA can be extracted from a healthy cell and one diagnosed with a disease (like cancer). After conversion of the RNA to cDNA, the fluorescence will be used to show the expression of genes in the cells in the body

What is RNA-seq?

Also known as whole transcriptome shotgun sequencing, RNA is isolated from a cell sample, and only that RNA is used, which will be converted to cDNA, alongside some linkers used. Useful to represent expression of genes due to how much mRNA molecules would’ve been present

What is northern blotting?

Uses denaturing gel to separate RNA to required size, then a highly specific probe is then used to detect whether a certain sequence of RNA is present

What is used to stop the sequencing of DNA?

Dideoxynucleotides, as they lack an OH for further dNTP’s to bind to

How many reactions can occur when a ddNTP is used?

4: ddATP, ddCTP, ddGTP, ddTTP

How are new strands of DNA visualised in a lab?

Fluorescent labels, strands that have been terminated by ddNTP’s and a lower background signal as to not pick up any enzyme activity that may have happened

What is next-gen sequencing?

Where multiple different sequencing technologies are used. DNA fragments are then sequenced in overlapping fragments which are then put back together to form a overall sequence

How are fragments of DNA identified?

DNA libraries have adaptors added to them using a slide. Clonal amplication by using a DNA polymerase, producing spots or beads with lots of single DNA fragments, so fluorescent signals are useful to identify the DNA fragments.

What is illumina NGS?

Where a slide of flooded with nuceotides and DNA polymerase, with every nucleotide having a fluorescent label, as well as a terminator, causing just one base to be added at a time. As each base has a different colour label, it is easy to determine which bases are which when an image is taken of the slide.

What is ion torrent / ion protein sequencing?

When dNTP is added to DNA, hydrogen ions (protons) are released, which decrease the pH of the buffer added. If a change in pH has occurred, then the base has been incorporated. The dNTP is washed away, then more is added, repeating the cycle multiple times.

What Long read 3rd gen single molecule DNA sequencing?

Solve the issues that illuma NGS have, like issues mapping repetitive elements, duplicated DNA sequencing and phasing alleles

What is high resolution melting?

Discriminates DNA sequences via nucleotide composition, length, GC content (Guanine and Cytosine) and strand complementarity.

What does allele specific oligonucleotide hybridisation do? (ASO)

Spots genetic variants via instabilities in hybridisation bonds between mismatched alleles. Amplifies the DNA via the use of PCR. It is then turned into ssDNA, which would be dot-blotted onto a membrane

What are oligonucleotides?

Small fragments of DNA, used for research and genetic testing

What is amplification refractory mutation system (ARMS)

Allele-specific oligonucleotide which uses PCR primers, and if the primer matches to the mutation, the DNA is amplified. If not, nothing happens.

What is taqman SNP detection?

Identifies single nucleotide polymorphisms. Uses primers to amplify DNA, allele probes which target specific alleles, probes labelled with different fluorescent dyes which determine whether each allele homozygous or heterozygous

What are single nucleotide polymorphisms

Genetic variants in just one nucleotide

What is single base primer extension?

A technique where ssDNA is targeted using a primer, binding to an area of interest, alongside a labelled nucleotide. Products are extended with the labelled nucleotide, which can determine mutations or alleles

How are biomolecules detected?

Using light

What is a chromophore?

A molecule that absorbs light, typically organic cyclic molecules or alkenes

What are some molecules that can act as chromophores, and are vital for body function?

Amino acids as well as proteins. Peptide bonds are also chromophores as well

How does ADH with alcohol cause a change in colour?

ADH converts alcohol into an aldehyde or ketone. This causes a rise in NADH, a change in colour, causes a spike in absorbance during reaction. Also shows changes in reactions too.

What is PAGE (Polyacrylamide gel electrophoresis)

Using electricity to separate proteins onto polyacrylamide gels

What is SDS (sodium dodecyl sulfate)-PAGE?

A detergent that denatures proteins. Beta-mercaptoethanol breaks disulphide bonds, causing them to lose their native state. Polypeptides coated with negative charges causing them to be separated based on molecular mass and size

What is isotonic point?

Proteins can be either basic or acidic (known as amphoteric molecules). The isoionic point therefore is where the protein has equal charges

What would happen to proteins at - or + pH differences

If pH is lower than the proteins isoionic point, then the protein will be + charged
If pH is higher than the proteins isoionic point, then the protein will be - charged
If pH is equal to proteins isoionic point, then the protein will be neutrally charged

What is isoelectric focusing?

A techinque which seperates proteins based on isoelectric points (where proteins don’t migrate to an electric field)

What’re the dimensions used during 2d electrophoresis?

Isoelectric point and molecular weight / protein mass

Is staining specific or non specific in terms of proteins?

Non-specific. All proteins can be seen

Ways to detect proteins after PAGE?

Staining, radioactive counting or immunoblotting (using proteins and antibodies)

What is the main benefit of silver staining during PAGE?

Incredibly sensitive in presence of proteins, detecting under a nanogram of protein

Detection limit of ethidium bromide when bound to DNA

0.5 to 5 nanograms per band

What is agarose gel used for?

Separates DNA based in basis of base pair length

Is blotting specific or non-specific?

Highly specific, as it only detects one biomolecule due to the use of antibodies

Different types of blotting

What are labels / probes?

Probes examine labels, which are molecules which can be seen through binding to a specific molecule

How does western blotting / immunoblotting work?

SDS-PAGE is used, where antibodies are used to attach to the specific protein wished to be detected

What is the area where an antibody binds to an antigen?

An epitope

Difference between primary antibody and secondary antibody?

Primary antibodies bind to antigen, and secondary antigen which binds to the primary antigen

Detection method for western blotting?

Radiolabelled (not used as often these days), chemiluminescence (film capture or imaging equipment) and fluorescent secondary antibodies

General properties of enzymes

Highly specific and very powerful, increasing reaction rates by over 100’s of thousands. Also decrease the energy pathway required for reactions, acting as catalysts. Also can be activated and inhibited via external factors

What are isoenzymes?

Enzymes which have very similar multiple forms to each other, but have slightly different amino acid sequences, with specific isoenzymes being produced in certain tissues.

What is proteolytic activation for enzymes?

Regulates apoptosis via the use of caspases, which are enzymes that cause cell death