theme 10: expression 3 : analysis of products.

Question 1

What is transcriptone? Why its important to analyse transcryprone in genetic engineering?

Answer 1

It is the complete set of RNA molecules, that are produced by a cell, tissue, or organism at a given time.

By studying the transcriptome, researchers can identify which genes are being expressed in response to specific environmental conditions, treatments, or genetic modifications.

Question 2

What techniques are used to determine how efficiently genes/PROTEINS were expressed?

Answer 2

1) RT-PCR (Reverse Transcription Polymerase Chain Reaction) (RNA)
2) Northern blot (RNA)
3) SDS-PAGE (protein) not a quantitative analysis
4) Pulse-chase technique (protein)

Question 3

Pulse chase technique. What does pull and chase stand for?

Answer 3

Pulse- label (adioactive or fluorescent) (for example 35S-methionine) is incorporated into synthesied molecules, like in this case a protein.
Chase- labeled molecule is tracked over time: folding, trafficking, modifications like cleavage are observed.

Question 4

What is fusion protein?

Answer 4

Combination of coding sequences from two different genes .

Question 5

Why affinity tags are added to proteins?

Answer 5

To increase protein solubility, to facilitate detection, purification.

Question 6

Name affinity tags?

Answer 6

Glutathion S-transferase (GST)- stabilizes protein, protects from proteolysis.
SUMO (Small ubiquitin-related modifier)- supports protein folding
Maltose-binding protein-used to increase solubility
His-tag protein- easy purification and detection of the target protein.

Question 7

What enzyme is used to decrease protein solubility?

Answer 7

ketosteroide isomerase

Question 8

What is the main purpose of intein?

Answer 8

During thiol-induced cleavage, intein cleaves itself from the target protein and leaves it tag free.

Question 9

What is intein-mediated protein trans-splicing? Where can it be used?

Answer 9

The inteins from 2 different proteins work together to ligate two domains, forming a new protein.
Can be used in chimeric proteins: two different protein segments are joined together. This is useful in creating proteins with combined functions or new properties that the individual components don’t have.

Question 10

Wat is epitope tagging?

Answer 10

Epitope: a short peptide sequence, which is recognised by a specific antibody, is genetically fused to a protein of interest. This technique is used for easy protein detection, purification.

Question 11

What is GFP and why its benefcial to use it?

Answer 11

reporter gene
GFP – Green fluorescent protein; exhibits fluorescence when exposed to light in the blue to ultraviolet range

Used for: finding localization of proteins within living cells ( For example, GFP can be fused to a membrane protein, allowing researchers to see the protein’s localization in the cell membrane.)

Question 12

Reporter genes-what are they used for?
What used with?

Answer 12

Are used with promoters; can measure the activity of a gene.

Question 13

Chloramphenicol Acetyltransferase (CAT). What is it and how it is used?

Answer 13

Its a reporter gene; It provides a quantifiable measure of gene expression by catalyzing the acetylation of chloramphenicol, rendering it inactive.

Question 14

What is Rabbit Reticulocyte Lysate (RRL)?

Answer 14

is a cell-free system used for in vitro translation of mRNA into proteins.
It is derived from the cytoplasm of rabbit reticulocytes, which are immature red blood cells that are rich in ribosomes and translation machinery.

Question 15

Heterologous proteins?

Answer 15

these are proteins that are produced in a foreign system

Question 16

What is wheat germ extract?

Answer 16

Wheat germ extract is widely used in in vitro gene expression systems to produce proteins from mRNA in a cell-free environment. It is derived from the germ of wheat seeds and contains a rich set of biological components, including ribosomes, translation factors, tRNAs, and other essential molecules for protein synthesis.

Question 17

Tumor inducing plasmid (Ti)?

Answer 17

Their ability transfer genes into plant cells
makes them Used for creating genetically modified plants with beneficial traits

Question 18

Why we need to reduce the amount of ACC in plants? What does ACC stand for?

Answer 18

to prevent the production of ethylene, which can be useful in agriculture to delay fruit ripening, extend shelf life.
ACC – 1-aminocyclopropan-1-carboxylic acid

Question 19

How to make plants resistant to pests?

Answer 19

Creating transgenic plants: Bt crops- pest resistant plants, which have been modified to express a protein from the bacterium Bacillus thuringiensis. This protein is toxic to specific insect pests like caterpillars, beetles, and flies. When the insect feeds on the plant, the protein disrupts its digestive system, killing the pest.