Processes to learn for bio Flashcards

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1
Q

Transcription

A
  1. Occurring in the nucleus(in Eukaryotes) in cytoplasm (in bacteria), DNA helicase unzips DNA, and RNA Polymerase binds to the promoter region initiating transcription
    1. RNA Polymerase runs along template strand in a 3’-5’ direction building a complementary template strand in a 5’-3’ direction, stopping once its hits a stop codon(UAG,UGA,UAA) falling off, leaving us with Pre-mRNA.
    (RNA processesing,occurs only in Eukaryotes)
    3.Methyl-g cap is added to the 5’ prime end of Pre-mRNA molecule and a poly-a-tail is added to the 3’ prime end
  2. Spliceosomes cut out introns and splice together exons leaving us with m-RNA.
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2
Q

Alternative splicing

A

Alternative splicing enables the same gene to produce different proteins.Splicing involves different exon splices where different exons are combined to form several kinds of mRNA, each with a different base sequence.

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3
Q

Translation

A

Initiation
1. The 5’ end of mRNA molecule binds to Ribosome and is read until the start codon (AUG) is recognised.Then a tRNA molecule with a complementary anti codon (UAC) binds to ribosome delivering the amino acid methionine,signifying the start of translation

Elongation-
2. After the first amino acid attaches, mRNA molecule is fed through the ribosome so the next codon can be matched to its complementary tRNA anti-codon.Complementary tRNA molecules deliver specfic amino acids to the ribosome, which bind to adjacent amino acids with a peptide bond via a condensation reaction.The first tRNA molecule then leaves the ribosome and continues to pick up amino acids, growing the amino acid chain

Termination-
3.Linking of amino acids in the polypeptide chain continues until the ribosome raches a stop codon.The stop codon signals the end of translation.The poplypeptide chain is then released by the ribosome into the cytosol or endoplasmic recticulum.

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4
Q

Trp operon regulation when levels are high

A

1.To regulate the expression of Tryptophan structural genes, the regulatory gene for trp operon is constantly expressed, producing a repressor protein

2.when levels of tryptophan are high meaning they are present. Tryptophan is able to bind with the repressor protein inducing a confromational change (physical change) in the repressor, allowing it to bind to the operator region.

3.This allows the repressor protein to bind to the operator region

4.Acting as a block, the repressor protein inhibits the transcription of the tryptophan structural genes by RNA polymerase, inhibiting the unnecessary production of tryptophan.

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5
Q

Trp operon regulation when levels are low

A

1.When Trypophan levels are low, there is not enough tryptophan molecules present that are able to consistently bind to the repressor protein,causing it too detach from the operator region.

3.This allows RNA polymerase to transcribe the trp structural genes so that level of tryptophan can increase

4.However, as tryptophan accumaltes in the cell it will once again bind the repressor protein slowly stopping transcritption of the trp structural genes

5.Together, these mechanisms keep the amount of tryptophan available at a relatively constant level to ensure that energy and resources are expended appropriately.

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6
Q

Trp operon by attentuation when levels are high

A
  1. Ribosome runs past the Tryptophan codons present in region 1 of the gene (since tryptophan is present)however, pauses between region 1 and 2 due to a stop codon

2.This prevents region 1 from binding with 2 and forces region 3 to bind with 4, forming a “hairpin loop”

3.This puts tension on the weak attenuator region (Due to adenine and thymine 2 weak hydrogen bonds), and mrna strand is forced too pull away ending transcription

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7
Q

Trp operon by attenuation when levels are low

A

1.Ribosome pauses at the 2 tryptophan codons in region 1 waiting for a tRNA molecule to bring tryptophan amino acid.

2.This allows 2 to pair with 3 preventing 3 and 4 from pairing with each other

3.while this creates a hairpin loop,its too far away from the attenuator region and therefore mrna doesn’t pull away and the ribosome continues transcribing the genes, creating more tryptophan

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8
Q

How are proteins secreted (full process)

A

1.Proteins intended for secretion are synthesised at the ribosomes found on the Rough Endoplasmic reticulum.

2.They are then folded at the Rough ER and are transported to the golgi aparatus, via transport vesiciles.

3.At the Golgi apparatus, the proteins are modified before being packaged into secretory vesicles.

4.These secretory vesicles fuse with the plasma membrane, releasing the proteins into the extracellular environment via the process of exocytosis.

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9
Q

Process of exocytosis

A

1.A vesicle containing secretory products is transported to the plasma membrane via secretory vesicles
2.The membrane of vesicle fuses with the plasma membrane
3.The secretory products are released from the cell into the extracellular environment

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10
Q

CRISPR-Cas9

A

Exposure:
Bacteriophague injects its viral DNA into bacterium,with cas 1 and cas 2 (endonuclease, enzyme that cuts DNA)cutting out the PAM sequence(protospacer region) of viral DNA.

Expression:
This Pam region then gets inserted between the repeater regions of the CRISPR sequence and is transcribed,becoming guide RNA

Extermination:
GRNA attaches to cas9 (endonuclease) which forms a Crisper-cas 9 complex.This is Guided to attack any renterring viral DNA since it has a complementary sequence.The next time viral DNA enters the bacterium, The CRISPR cas-9 complex identifies the viral DNA and the cas 9 cuts sugar phosphate backbone of the viral dna, inactivating the virus.

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11
Q

CRISPR-cas 9 in gene technology

A

1.Synthetic gRNA is made in a lab containing the complementary sequences to the target gene that scientists wish to cut ,Cas9 an endonuclease with an appropriate pam sequence is then identified

2.Synthetic gRNA is then mixed with cas-9 to form the CRISPR-CAS9 complex,this formation is then injected into a specific cell such as a zygote

3.The Cas9 finds target pam sequence and using the grna, checks to make sure it has complementary sequence

4.Cas 9 works and cuts at the restriction sites leaving blunt ends,Scientists can then introduce new nucleotides hoping it will ligate back into the DNA

5.DNA attempts to ligate back together

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12
Q

PCR

A

Denaturation:
DNA is heated to approximately 90-95c causing it to denature,breaking the hydrogen bonds between the bases to separate the strands,forming a single stranded DNA.

Annealing:
The single stranded DNA is cooled to approximently 50-55c to allow the primers to bind to complementary sequences on the single-stranded DNA

Elongation:
The DNA is heated up again back to 72c which allows taq polymerase to work optimally. Taq polymerase binds to the primer which acts as a starting point and begins synthesizing a new complementary strand of DNA

Repeating:
steps 1-3 are repeated multiple times to create more copies of DNA

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13
Q

Gel Electrophoresis

A

1.Dna is cut by restriction endonucleases or a short sequence of DNA that has been amplifieid during PCR

2.DNA samples are placed in wells within an aragose gel (Negative side),The aragose gel has tiny pores that allow gel to pass.The aragose gel has been immersed in ion rich buffer allowing an electrical current to pass through, a standard ladder is also added to determine length.

3.An electrical current one positive and one negative is passed through Gel.Since DNA has a negative charge (due to its phosphate backbone), it moves towards the positive end.

4.Smaller DNA fragments move faster through the Gel and travel further than larger fragments, after a few hours current is switched off , as DNA fragments stop moving they settle into bands

5.DNA is difficult to see so its stained with a florouscent dye such as ethidium bromise which allows bands to be viewed under UV lamp.

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14
Q

Recombinant plasmid (Circular DNA vector that is ligated to incorporate a gene of interest, edited to integrate a target gene)

A
  1. Gene of interest is isolated and copied using PCR

-The Gene of interest cannot have introns prior to insertion,since prokaryotes dont have introns and therefore bacteria wouldn’t know what to do with intron segments,Introns are typically excluded from the gene of interest via two different methods; synthetic DNA, which is made in a lab by scientists without introns and cDNA (copy DNA) which is made by a an enzyme called reverse transcription which functions to transcribe mRNA backwards into cDNA

  1. Plasmid vector is selected into which gene of interest will be inserted

Plasmid vectors contain 4 essential parts
- Antibiotic resistance genes- (e.g ampR,tetA) This antibiotic resisitance that the plasmid vector allows it too be seen even after the antibiotic is added to the recombinant plasmid later on
- Origin of replication (ORI)-Sequence that signals the start site for DNA replication in bacteria
- Reporter gene- Genes that have an easily indetifiable phenotype, that a

  1. DNA of plasmid is cut at one point by restriction endonucleases, that create sticky ends, This changes the form from circular to linear.
  2. The gene of interest is then cut using the same restriction endonucleases that cut the plasmid, too ensure that the plasmid and gene of interest have complementary sticky ends
  3. The gene of interest (Foreign DNA fragments) and plasmids are then mixed and in some cases, their sticky ends pair by weak hydrogen bonds, the complex is now a recombinant plasmid
  4. The plasmid may not accept the pairing and therefore just reseal without the foreign DNA transforming back to just a plasmid, however if the Plasmid accepts,Ligase is added and this makes the joining permanent through covalent bonding.

antibiotics can be used to identify whether a plasmid has taken up the gene of interest

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15
Q

How to encourage plasmid to accept inserted gene

A

Heat shock:Using temperature to increase permiability of plasmid membrane:
1. Bacterial culture is placed into ice bath ,Recombinant plasmids with the antibacterial resistant allele (i.e tetR) are added to bacteria culture which is chilled
2. The bacteria and plasmid is then heat shocked by being placed into hot water around 42c for 50 sec, this heat shock causes the plasma membranes bacterial cells to alter, increasing the chance of the cell uptaking the plasmid
3. The mix is then returned to ice bath to return plasma membrane to natural state
4. The bacteria is then plated on a agar plate along with antibiotic (i.e tetracyline) this is then incubated at 38c overnight.
5. Bacteria that have no taken up the plasmids will be killed by the antibiotics but bacteria that have taken it up will not since they inhabit the antibiotic resistant alleles from the plasmids.
6. Bacterial cells that have taken up the plasmids will be selected and transformed, replicating

or

Electroporation:
1.Electrical current is passed through solution containing plasmid and bacteria,Electrical current causes the plasma membrane to become more permeable allowing plasmid vectors to cross through the plasma membrane

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16
Q

Insulin production

A

1.Plasmid vector with ampR and tetR gene for resistance is produced.

  1. Insulin A and B subunit genes are cut using restiction endonucleases (EcoRi and Bamhi) too form sticky ends and recombinant plasmids.DNA ligase is added too reform, the sugar phosphate backbone of the DNA creating transformed bacteria.
  2. Plasmids are added to solution of E.coli bacteria and then either heat shock or electroporation is used to increase the uptake of the plasmids in the bacteria.

4.To identify which bacterial colonies took up bacteria it is added to agar plate with ampR those that survived may have been bacteria.Second test with tetR is used, those that die have the recombinant plasmid since the insertion of insulin subunit gene disrupts the tetR resistance gene.These plasmids are then collected.

5.Plasmids are cut using Ecori again to insert lacz (report gene) into plasmid.LacZ produces b-galactodase.Plasmids containing lacZ are then added too Ecoli bacteria to create transformed bacteria.

6.Lac-z produces B-galactodase which can create a blue coloured compound cause of x-gal.This means colonies that were blue could be identified to have taken up recombinant plasmid.These bacteria are able to produce insulin subunit gene which is attached to B-galactodase.

7.Transformed bacteria that contains the recombinant plasmid are then placed into conditions to reproduce before their membranes are broken down and the human insulin that has been produced is isolated and purified.The two insulin chain have their b-Galactosidase tails removed and are mixed together, which allows the connecting disulphite bonds to form and create functional human insulin.

17
Q

How to produce transgenic plants

A

Gene identification:
- Gene of interest must be identified and isolated

Gene delivery:
-Isolated gene of interest is delivered into the cells via the host organism. This delivery may occur either via,direct insertion of DNA into the genome of the plannt itself, or through the use of bactieral plasmid that is able to transfer DNA between itself and the plant

Gene expression:
-transformed cell is grown repeatedly, using plant tissue cultures under sterile conditions before being applied in the feild for agricultural use

18
Q

Steps of the light-dependent stage include the inputs and outputs

A

Occurs in the thylakoid membranes

1.Light excited electrons within the chlorophyll,water previously absorbed splits into oxygen and hydrogen donating an electron to chlorophyll

2.The movement of hydrogen ions (Protons) down its concentration gradient, allows for the formation of coenzyme proton carriers NADPH and ATP

4.Oxygen becomes an output and is diffused out the plant via the stomata, or it is used as an input of aerobic cellular respiration.

5.ATP and NADPH (coenzymes) move to the stroma, where they are used for the light indpenedent stage.

Inputs
-Sunlight
-h2o (water)
-ADP+Pi
-NADP

outputs
-02
-ATP
-NADPH

19
Q

Light-independent steps include inputs and outputs

A

(occurs in the stroma)

Carbon fixation
Carbon dioxide enters the calvin cycle via the stomata.Carbon fixation occurs as it binds with RuBp to form 3-PGA

Reduction
NADPH molecules formed in light dependent reaction donate their hydrogen ions and electrons, ATP breaks down into ADP + Pi these reactions release energy facilitating further changes with the glucose molecules.A specific G3P carbon molecule is formed which then goes on to contribute to the formation of glucose

Regeneration
Some oxygen molecules left over from reaction combine with hydrogens to form the output of water.NADPH and ATP are then recycled back into the light dependent stage.

Input
-carbon dioxide
-ATP
-NADPH

Output
-Glucose
-ADP+pi
-NADP+

20
Q

Rubisco process in c4 plants

A

(Occurs in mesophyll cell)
-Carbon dioxide (CO2) enters mesophyll cells and is fixed by the enzyme pep carboxylase, the CO2 molecule binds with the three carbon molecule (PEP) and forms a 4 carbon molecule (oxaloacetate),(pep carboxylase enzyme responsible for carbon fixation in c4 plants has no affinity to bind with O2)
-Oxaloacetate is then converted to the four carbon molecule malate and is transported to bundle sheath cells

(occurs in bundle sheath cells)
-inside bundle sheath cells, malate breaks down and releases CO2 -Carbon dioxide enters the calvin cycle in exactly the same way as c3 photosynthesis
-pyruvate is then formed from the breakdown of malate and is transported back to the mesophyll cell and converted to another molecule, PEP, with the help of ATP
-PEP is then ready to contribute to the fixation of CO2 and production of oxaloacetate and the cycle continues all over again

21
Q

Rubisco in CAM plants

A

-At night CAM plants open up there stomata to bring in CO2, the CO2 is then fixed into a 4-carbon molecule (oxaloacetate) by the enzyme PEP carboxylase

-Oxaloacetate is then converted to malate which is stored inside vacuoles within the mesophyll cell until the daytime

-During the daytime, CAM plants dont open their stomata to prevent water loss

-Malate is transported out of the vacuole and broken down to release CO2

-CO2 is then free to enter the calvin cycle.

22
Q

Glycolysis (name inputs and outputs)

A

First stage of aerobic cellular respiration,occuring in the cytosol of the cell, in this stage glucose is converted to 2 pyruvate molecules.Glucose a carbon six molecule is broken down via a sequence of enzyme regulated reactions to form 2 pyruvate molecules (two three-carbon molecules)

Inputs
-1 glucose
-2 ADP + 2P
-2 NAD+ 2H

Outputs
-2 pyruvate
-2 ATP
-2 NADH

23
Q

Krebs cycle

A

Occuring in the matrix of the inner compartment of the mitochondria. Pyruvate is transported to the matrix of the mitochondria and combines with coenzyme A to form acetyl-coas, this reaction releases carbon dioxide and produces NADH.

Inputs:
-2 acetyl-CoA (gained from the 2 pyruvate)
-2ADP + 2P
-6NAD + 6H
-2FAD + 4H

Outputs:
-4 carbon dioxide
-2 ATP
-6NADH
-2FADH2

24
Q

The electron transport chain

A

Occurs in the cristae of the mitochondria, process In which a series of protein complexes embedded in the inner membrane of a mitochondria harness the stored energy in NADH and FADH2, to generate large amounts of ATP.
Aerobic cellular respiration produces 30 or 32 ATP molecules per glucose molecule.This is why its essential for humans to breath oxygen, so they can create lots of energy.

Inputs:
-6 oxygen (o2) + 12 H
-26 or 28 ADP + 26 or 28 pi
-10 NADH
-2 FADH2

Outputs
-6 water (h2o)
-26 or 28 ATP
-10 NAD + 10 H
-2 FAD + 4H

25
Q

Biofuels process

A

Made from biomass via the process of fermentation.
Deconstruction of the original biomass:
-Biomass is treated causing it too breakdown increasing its surface area,making the fermentation process more efficent.

Enzyme catalyzed hydrolysis of complex sugars:
-Starch and cellulose are converted into glucose and other sugars.This is aided by the presence of water in a process known as hydrolysis.

Anaerobic fermentation to produce ethanol:
-Yeast is used to facilitate the anaerobic fermentation of the sugars produced in step 2,large amount of ethanol is produced.ethanol diffuses out of the yeast and is harnessed for biofuel

Final distillation and purification of the ethanol for use of fuel:
-Ethanol is distilled via the removal of water converting It into a useable form called biofuel.

26
Q

Phagocytosis

A

(engulfing and destroying of pathogens by phagocytes like dendretic cells or macrophages,or otherwise known as antigen presenting cells)

1.Pathogen enters the body
2.pathogen fuses with lysozome
3.Lysozome releaes ensymes that begin to degranulate pathogen
4.Pathogen is brocken down into small gfragments
5.fragments are displayed on antigen presenting cells surface

27
Q

Antigen Cell Presentation:

A

1.After Phagocytosis APCs (Macrophages and dendretic cells) that display the pathogens antigens on its surface travel to lymph nodes via the lymphatic system
2.Here, T helper cells use specialised proteins called CD4+ which recognise the shape of antigen,finding a complementary T helper cell
3.Once a T helper cell is found it secretes cytokines to create many more T helper cells to help identify which active pathway it will undergo

28
Q

Humoral response

A

(occurs in bodily fluids such as blood)
1.B cell with complementary shape to pathogen is identified and is said to be “Selected”
2.T Helper cell selected in antigen presentation releases cytokines intiating clonal expansion of B cell and differentiation
3.B cell differentiates into B memory cells vital for longlasting immunity and B plasma cells which secrete antibodies
4.Antibodies specific to pathogen are released

29
Q

Cell mediated response

A

(occurs in infected body/skin cells)
1.Naive T cell with complementary shape to Pathogen is identifed and selected
2.T helper cell selected in antigen presentation stimulates clonal expansion of Naive T cell via the release of cytokiness
3.Cytokines released also stimulate differentiation where T memory cells via to long lasting immunity are made and cytotoxic T cells are made
4.Cytoctoxic T cells leave lymph nodes travel to site of infection initiating apoptosis via the release of chemicals like preferen

30
Q

Monoclonal antibodies production process

A

Lab produced antibodies made to specifically bind to antigen

1.Scientists identify and isolate antigen present on target cell ,typically it is the antigen causing disease that is selected.

2.Via vaccination antigen is given too mouse, who undergoes a immune response producing a B lymphocyte that matches the antigen, scientists extract this B lymphocyte from the Mouse spleen

3.B lymphocyte is then removed and fused with myeloma cells to frorm a hybridomen.Myeloma are chosen over vitro because B lymphocytes do not grow well in vitro whereas they do in myeloma which gives them the ability to grow alot and produce large quanities of anitbodies.

4.Hybridomas with appropriate antibodies are chosen and cloned resulting in mass antibody production

5.Antibodies are collected and purified given back to the patient

31
Q

Inflammatory response

A

Intiation:
In response to injury macrophages situated in tissue become activated and along with damaged cells release cytokines.Additionally mast cells present degranulate and release histamine.

Vasvodilation:
Histamine released from mast cells travel to nearby blood cells and bind to specific receptors causing vasvodilation.this causes blood cells to widen increasing blood flow, this is what causes the swelling,redness and warmth.Additionally the formation of gaps in the vessel wall increase its permeability to the cells of the immune system.

Migration:
Vasvodilation and the increased leakiness of blood cells allow for a number of innante immune system cells to leave the blood stream and enter the site of injury.Some of these cells include; Phagocytes that phagocytise pathogens and complement proteins that attract pathogens and make it easier for phagocytes to destroy them.

32
Q

Types of immune cell therapy

A

Dendretic cell therapy:Involves the priming of dendretic cells (phagocyte and APC) with tumour associated antigens (TAAs) to facilitate the activation of lymphocytes (B and T cells), priming hem to kill any cells with the tumour antigen.This priming can be achieved by the vaccination of TAAs or removing dendretic cells from body and priming them with TAAs before infusing them back into patient.

CAR-T therapy:Involves the modification of T cells to recognise and destroy cancer cells.t cells are extracted form patient and scientists add a gene coding for an antigen receptor to its DNA.This protein then gets synthesised by the cell and inserted into its membrane allowing it to recognise cancer cells.These cells now have Chimeran antigen receptors and are inserted back into the patient.

Antibody therapy:Creation and use of antibodies to stimulate and enhance the function of the immune system.Antibodies used in antibody therapy are typcally monoclonal antiboides.

Cytokine therapy:Involves the use of immune signalling molecules like interferons and interleukins to modulate the effect of the immune system.