Biology Unit 3 SAC 1 Flashcards
Restriction Endonuclease
Type of enzyme that acts like the molecular scissors, cutting nucleic acids strands at recognition sites (AKA Restriction enzymes)
Endonuclease
Type of enzyme which breaks phosphodiester bonds between two nucleotides in PPC
Ligase
Enzyme that joins two molecules together by catalysing formation of phosphodiester bonds
Polymerase
Enzyme that synthesis polymer from monomer such as DNA strand from nucleic acids.
Primer
Short single strand of nucleic acid that acts as starting point for polymerase enzyme to attach on
Explain the 3 steps of CRISPR-Cas9 in bacteria
1) Exposure - The bacteriophage injects its viral DNA into the bacterium which is recognized as foriegn. Cas1 and Cas2 recognizes the PAM sequence which signals to extract protospacer from viral DNA enzymes work together to cut a section of the viral DNA just before the PAM and introduces it to the bacteriums CRISPR gene becoming a spacer.
2) Expression - The CRISPR spacers are transcribed and converted into RNA molecules known as gRNA. the gRNA binds to the cas9 to create CRISPR-Cas9 complex which is directed to any viral DNA inside the cell that is complementary to the gRNA
3) Extermination - CRISPR-Cas9 complex brouses cell for other invading bacteriophage complementary to gRNA sequence and when it does the Cas9 inactivates the virus through the phosphate-sugar backbone. Cas9 contains 2 active sites which cuts both strands of DNA and creates blunt ends.
Explain the 8 steps of CRISPR-Cas9 in gene editing
1) Synthetic sgRNA is created in lab which has complementary spacer to target DNA scientists wish to cut/silence
2) Cas9 enzyme obtained with appropriate PAM target sequence
3) sgRNAand Cas9 are put in a mixture together to bind creating CRISPR-Cas9 complex
4) sgRNA and Cas9 mixture is injected into specific cell such as Zygote
5) Cas9 finds PAM target sequence and check if sgRNA aligns with DNA
6) Cas9 cuts selected sequence of DNA
7) The DNA has blunt end which the cell will try to repair
8) When repairing the DNA the cell may introduce new nucleicotides into DNA, and scientists may inject particular nucleotide in hopes it will ligate the gap
Applications of CRISPR-Cas9 in RESEARCH
- Attaching fluorescent protein to Cas9 to locate specific gene in genome.
-Disrupting the expression of genes to see the effect of that protein being knocked out. This helps scientists identify the function of specific genes.
What makes CRISPR unique as restriction endonuclease in a tool for human research
CRISPR is unique as an endonuclease because it can be ‘programmed’ with different g/sgRNA molecules to cut at different sequences, unlike other endonucleases which can only cut at one specific recognition site
(e.g. the restriction endonuclease SmaI only cuts at CCCGGG, whereas CRISPR can be programmed to cut at any desired sequence by changing the gRNA/sgRNA molecule).
Why CRISPR-Cas9 should be inserted into a zygote to produce a change for whole oragnsim
To produce changes throughout the whole oragnsim it firstly starts of with the zygote the first cell of a new oragnsim because the zygotes DNA is passed down to all subsequent cells, as we are too developed now.
What is the overall structure of DNA and RNA with reference to the bonds
DNA is double stranded helix, each strand with a backbone of sugar-phosphate linked by covalent bonds called phosphodiester bonds. The complementary base pairing (A, T, C, G) are held by hydrogen bonds.
RNA is single stranded and instead of a Thymine base it contains ucrail instead.
Name and define 5 bioethical concepts
1) beneficence - the commitentment to maximising benefits, and minimsing risks
2) integrity - the commitment to searching knowledge and understanding. the honest reporting of all sources
3) respect - the acknowledgement of intrinsic values of all living things, and the
4) justice -
5) non-malefience - non harm
name and define the 3 bioethical approaches
1) consequences based - the aim to maximise positive outcomes and miniside negative outcomes
2) duty-rule based - Promotes responsiblity of the agent and importance of duty
3) virtues based - Emphaises indivduals goodness and promotes acting in accordance with values of moral person
What is the purpose of PCR?
The purpose of PCR is to amplify a specfic segment of DNA by making multiple identical copies, used by scientists when there is insufficient amount of DNA sample.
- Forenstic testing
- Paternity testing
- Analysing gene fragments for genetic diseases
Name and explain 3 steps of PCR with temperatures
1) Denaturation - DNA is heated to 90-95 degrees to break hydrogen bonds between bases forming single stranded DNA
2) Annealing - Single stranded DNA is cooled to 50-55 degrees to allow primers to bind to complementary sequences
3) Elongation - The DNA again is heated to 72 degrees allowing Taq polymerase to work optimally binding to the primer and begins synthesisng new complementary DNA strand
4) Repeat the cycle multiple times for multiple copies.
Gel electrophoresis
Technique to separate DNA fragments based on their molecular size.
The set of gel electrophoresis:
- Wells: Indent in gel into which a DNA sample is loaded
- Standard ladder: mixture of DNA fragments of known length that are used to infer the size of fragments in a sample
- Buffer solution: Ion rich solution which carries electrical current through agarose gel.
-Porus Gel: The gel has porous (holes) in them - Electrodes: One positive and negative electrode, negative positioned near Wells and positive opposite end of gel. Since DNA is negatively charged it will be attracted to the positive electrode. When the electrical current is applied the DNA fragment will move through the pores towards positive.
How the size DNA fragments affect their movement through the gel
The smaller fragments move furthur to the positive electrode due to gel agarose having smaller pores allowing for the movement of DNA. (faster and furhur)
Larger fragments move slower due to being to large to pass through the pores of the agarose gel.
Plasmid
Small circular loop of DNA found in bacteria
Vector
Way of introducing foriegn DNA into oragnsim (plasmids being a popular vector in bacterial transformation)
Bacterial Transformation
The process where bacteria takes up foreign DNA and scientists use this process to introduce recombinant plasmids into bacteria
Recombinant plasmid
Small circular loop of DNA vector ligated to include a gene of interest
Components of plamid vector
-Antibiotic resistance gene: gene which confers antibiotic resistance
-Orgin of replication: sequence found in prokaryotes signalling start site of DNA replication
- Reporter gene: Gene with easily indentifable phenotype used to identify whether a plasmid
has taken up the gene of interest.
Explain RNA processing
The modification of pre mRNA to mRNA used in translation.
1) Alternative splicing - Removal and rearranging of exons to give rise to many different mRNA strands coding for different proteins.
2) Addition of 5-methyl gap cap 3-poly A tail - Serves to stablise mRNA molecule preventing it from degrading and allowing it to bind to ribsome during translation.
Genetically modified organisims (GMO)
Organism with genetic material that has been altered using genetic engineering technology.
Transgenic organsim
GMO containing foreign genetic material from a separate specie.
Producing genetically engineered plants with reference to increasing crop productivity
1) Gene Identification - Gene of interest is identified and isolated
2) Gene delivery - Isolated gene is delivered into the cells of host organsims
3) Gene expression - Transformed cell is then grown repeatedly using plant tissue cultures and then the GM is able to express the new transgene as useful protein and can regenerate itself
Describe Transcription
The RNA polymerase binds onto the promotor region signalling hydrogen bonds to weaken and causing the DNA helix strands to unwind/unzip. The RNA polymerase continues down the template strand using free-floating complementary RNA nucleotides to produce a single strand of pre-mRNA until a termination sequence is reached ending transcription
Describe Translation
mRNA exits nucleus and the 5’ end of the mRNA binds to the ribosomes. tRNA molecules with complementary anticodons delivers amino acids. The mRNA is fed through the ribosomes so the next codons are complementary to the tRNA anticodon. tRNA delivers more amino acids to continue to grow the amino acid chain through condensation reaction. A STOP codon is reached ending translation PPC is released
Name biological, social, and ethical implications
Biological PROS
-GM crops have better crop productivity then non-GM crops
-GM foods can be made to improve nutritional content improving health of people
CONS
-GM crops may lose their effectiveness
-Widespread use of GM crops could result in loss of genetic diversity
Social PROS
-Increased crop productivity means more food produced
-GM foods can be made to improved flavour, texture giving ppl more appealing product
CONS
-Having to buy new seeds each seasons costly
-Complex legal issues regarding GM products = stress, anxiety to farmers
Ethical PROS
-Some believe using GM products is a ethical imperative giving benefits
CONS
-Some consider GMO unatural
-Some people GM foods are unsafe to eat
Name the 3 types of RNA
1) rRNA ribosomal RNA is the main strucutural component of ribosomes
2) mRNA messenger RNA sends genetic info from nucleus to ribosomes for protein syntheisis
3) tRNA transfer RNA delivers amino acids to ribosome after recognizing specific nucleotide sequences on mRNA
Name the 2 methods to allow plasmid to enter bacteria
1) Heat Shock: Bacteria and plasmid placed in calcium ion solution which is heated to 37-42 degrees for 25-45 seconds before being returned to ice making plasma membrane more permable
2) Electroporation: Electric current passes through solution containing bacteria, plasmid vectors making plasma membrane more permable.
Steps for RECOMBINATION
1) Gene of interest and plasmid are both cut with same restriction endonuclease to create identical sticky ends on either end of DNA sequence
2)DNA ligase will join gene of interest with plasmid vector creating recombinat plasmid.
3)The plasmid is inserted into bacteria through heat shock or electroporation
Steps for TRANSFORMATION
To distinguish between transformed and not transformed bacteria the mixture is placed on antibiotic rich plate
- Untransformed bacteria will be killed off when exposed to that antibiotic meaning each colony visible on plate represents transformation event
-If segment of DNA glows under UV light it means transformed bacteria has not took up recombinat plasmid
-If segment of DNA does not glow under UV light it means transformed bacteria took up recombinat plasmid
Name 4 properties of genetic code
1) Universal - All living organisms use same genetic code to code for specific amino acids
2) Umamigious - Each codon codes for specific type of amino acid
3) Degenerate - Amino acid may be coded by multiple codons
4) Non overlapping - Triplet/Codon read independently
Factors affecting gel electrophoresis
1)Voltage - the stronger the electric force the further DNA
travels towards the positive electrode
2) Gel composition - gels with greater density/agarose concentration increase difficulty for larger fragments to move through
3) Buffer concentration - the greater the concentration of ions in the buffer the more the
electric current is conducted through the gel
4) Time -the longer the electric current is applied, the further the DNA will travel.
Note: if too much time passes, the DNA may move out of the gel.
High levels of trp operon REPRESSION
Tryptophan binds to repressor proteins inducing conformational change in repressor protein changing active site.
This allows repressor protein to bind onto operator region.
In this way repressor protein prevents transcription of structural genes by blocking path of RNA polymerase inhibiting production of tryptophan.
Low levels of trp operon REPRESSION
Insufficeint levels of trp operon causes repressor proteins to become inactive and deattach from operator region allowing RNA polymerase to transcribe the structural genes.
Draw prokaryotic and eukaryotic gene
DRAW on whiteboard with 1-2 minute timer
Bioethical concepts in relation to GMO
Integrity - Manufacturs need to clearly label their products as GMO
Justice - GMOs might create inequity to larger companies who have resources to genetically alter their crops versus a small family farm that does not
Beneficence - Positive health outcomes for people consuming GMOs
Non Harm - GMO might cause disruptions to the food web
Respect - Important to promote the rights of individuals to freely chose if they consume GMO or not
Limitations of CRISPR-Cas9
Successful applications in animals, not humans
Cutting is precise but difficult to achieve precision when inserting DNA into a cell
Editing DNA in wrong place
Illegal to inject GM embryos
Embryo cannot consent to genes being edited
Costly procedures only available to wealthy people
Threat to those who society judge as biologically inferior
