Lab Exam 2

Question 1

What we are looking for when completing Amylase Experiment

Answer 1

• We are looking at the hydrolysis of starch catalyzed by amylase, which is a breakdown through hydrolysis reaction, producing maltose
  
  • Starch if left alone will hydrolyze on its own but with amylase we are speeding up the process
• all for the goal of determining the optimal temperature of the type of amylase enzyme we use

Question 2

Purpose of Maltose Standard Curve

Answer 2

to estimate maltose concentration of an unknown substance by knowing the absorbance level

Question 3

Procedure for Amylase Lab

Answer 3

1. predetermined amounts of Starch and water added and mixed(amounts are controlled for across all tubes) before put into their respective temperatures for 5 mins
   
   2. Amylase is then added while tubes are in their temperatures
   3. Incubate 10 mins to allow for amylase to catalyze hydrolysis of starch
   4. All tubes are taken out and 1000ul of DNS assay added
   5. Boil for 5 mins to
   6. Dilute with 8.0ml of deionized water
   7. place in spectrophotometer and read absorbance level at 540nm
      
      8. Figure out what absorbance level equals in maltose concentration using standard curve and divide by 10 mins to get amylase activity

Question 4

Cell Competence

Answer 4

the ability of DNA to cross a cell membrane

• as DNA backbone carries a negative charge and the bacterial membrane has an overall negative charge, the buffer of calcium chloride is added to neutralize the charges and increase competence

Question 5

Transformation

Answer 5

the process by which bacteria uptake these plasmids

Question 6

Process of Transformation

Answer 6

○ This is done by chilling the temperatures at 4 degrees in ice before heat shocking it and transferring it to a water bath for 90 secs at 42 degrees
○ This temporarily creates a hole in bacterial membranes allowing the plasmid to cross
○ Once it crosses we place it back in the ice bath to recover and fix the holes in the membrane
○ After a period of recovery, we then provide the bacterial cell with growth media(spreading it on Agar with LB broth) and incubate it at 37 degrees in order to give it the best possible environment to grow and survive

Question 7

Plasmids

Answer 7

extra chromosomal, self-replicating, circular loops of DNA which hold additional genes that bacteria don’t naturally have
- The plasmid in this experiment is a pGLO plasmid

Question 8

Components of pGLO Plasmid

Answer 8

○ Plasmids contain Ampicillin resistance gene(Bla gene which codes for Beta-Lactamase)
○ Also contains GFP gene which codes for a green fluorescent protein
○ Further has a araC gene which codes for the control of GFP expression(on off switch)
§ Doesn’t have araC on the actual plasmid as it is in the growth media
○ Has pBAD which is a promotor gene
-Origin of replication: allows for self-replication (binary fission)

Question 9

Antibiotic and Antiobiotic Resistance Gene definitions

Answer 9

Antibiotics: a drug which should inhibit bacterial growth and prevent bacterial infection if the bacteria is susceptible

Antibiotic resistance(AmpR): when bacteria acquires genes which produce an enzyme allowing it to evolve and break down antibiotics and survive

Question 10

Transformation Prac Experimental Procedure

Answer 10

• taking a sample of E.coli bacteria which are all susceptible to ampicillin
  
  • We then add in a solution containing plasmids with the Ampicillin resistance gene and GFP protein
    -got cells to undertake transformation through heatshock and freezing, conferring the pGLO plasmid to some of the cells.
• we then took samples of the bacteria, swabbed them onto an agar plate with growth median and antibiotics to see whether they would survive.
• we then tested the effectiveness of transformation by shining a UV light over the bacteria, and whichever bacteria are fluorescent, we can assume that they have up taken the plasmid due to them glowing because of the GFP gene in the pGLO plasmid

Question 11

How AraC Controls GFP Expression

Answer 11

• Arabinose is a sugar added in the median
  • Without arabinose, the switch is off meaning araC blocks RNA polymerase from binding to the promotor
    
    • With arabinose, the switch is on and changes shape, allowing polymerase to bind to the pBAD promoter and transcribe GFP gene

Question 12

Results of Bacterial Transformation

Answer 12

Plate A:
- arabinose, pGLO plasmid and water
- Exhibited growth
- Showed green glow

Plate B:
- water, ampicillin, arabinose
- Exhibited no growth
- No green glow

Plate C:
- water
- Exhibited lawn growth
- No green glow

Question 13

Purpose of each plate in Bacterial Transformation

Answer 13

Plate A: experimental plate

Plate B: control to show bacteria as susceptible to ampiclin without the amp resistance gene from pGLO

Plate C: control for bacteria not being killed throughout transformation process

Question 14

PCR

Answer 14

the selective amplification of target DNA
- Requires a small sample of DNA and DNA/Taq polymerase(Thermus aquaticus)
- Carried out in a thermo-cycler at 3 different temperatures(95,
- 2n copies of DNA made (N is the number of cycles)

Question 15

Steps in PCR

Answer 15

1. Denaturation: DNA is heated to 95 degrees for 30 secs to separate DNA into single strands by breaking hydrogen bonds
   2. Annealing: Lowered to 55 degrees allowing the primers to bind to the complementary sequences on Single DNA template strand
      3. Extension: Temperature raised to 72 degrees, which is taq polymerase optimal temperature, and allows for annealed primers to extend through synthesis of the complementary DNA strand

Question 16

Molecules/chemical elements required for PCR

Answer 16

• DNA template from each family member
  • The Reaction Mix which has:
    ○ DNA polymerase
    ○ buffer(for optimal polymerase activity)
    ○ DNT (Deoxynucleotide triphosphate) which holds all nucleotides(dA, dT, dC, dG)
    - Primers: short sections of DNA which help select which segment of DNA we want to replicate

Question 17

Background information on Hemoglobin and Sickle Cell Disease

Answer 17

• Hemoglobin is made up of 4 subunit polypeptides
  
  • Sickle cell disease is a recessive disease caused by a mutation in the HBB gene coding for beta globin, one of the two polypeptide chains that form hemoglobin
    ○ HbA codes for the normal trait of hemoglobin
    ○ HbS codes for the sick trait of sickle cell disease
  • It is caused by a single nucleotide change in the hBA gene converting glutamic acid to valine
  • As valine is hydrophobic, it makes HbS molecules aggregate and clump, causing them to form abnormal sickle shape cells,
  • This reduces blood cell count as well as makes it more difficult for blood cells to flow through small capillaries

Question 18

Purpose of PCR Lab

Answer 18

• A family has a history of sickle cell
  
  • Job is to test each family member to see whether they have the disease, they are a carrier or whether they are normal
  • Our target DNA is a segment of the gene coding for beta-globin, a subunit polypeptide

Question 19

What is purpose of gel electrophoresis in the PCR experiment?

Answer 19

By pairing it with a restriction endonuclease which cuts at a specific site on normal polypeptide chains, if allows us to see how many different bands are created, which allows us to see the different lengths of strands resulting from the activity of the endonuclease, and therefore whether the individual has the disease, is a carrier or is normal and unaffected.

Question 20

Restriction Enzyme and its intended effects?

Answer 20

Restriction enzyme used: Dde1

- For homozygous normal we expect two fragments
- If heterozygous, we expect 3 fragments
    - If homozygous recessive we expect 1 fragment

Question 21

Gel Electrophoresis

Answer 21

• A method of separating molecules based on size and charge
  • Agarose gel is a pore-ish gel and DNA is loaded into the wells at one end
    
    • A electrical current is then sent from the wells side, and because DNA is negatively charged, it will move away from the wells towards the positive end of the gel, through all the pores

Question 22

How we get exact BP measurements of sample DNA in gel electrophoresis

Answer 22

we add a DNA ladder which comes with known DNA sizes, into the gel and therefore can compare these known results with our samples to estimate bp length

Question 23

Expected Results of Gel Electrophoresis

Answer 23

• If the DNA sequence is normal and contains the HbA sequence, restriction endonucleases will recognize its sequence and cleave the DNA into two smaller pieces
  
  • This will allow it to travel further during gel electrophoresis
  • If the DNA sequence has mutated and contains the HbS sequence, the restriction endonuclease will not recognize a sequence and therefore won’t cut the DNA, leaving it big.
    This means during gel electrophoresis, it will not travel as far due to getting stuck.

Question 24

Procedure for Endonuclease reaction and Gel Electrophoresis

Answer 24

1. Prepare reaction mix to go into 4 tubes(adding and spinning to mix)
   
   2. Add a sample of each person’s DNA into 1 of each tube
   3. Spin all 4 tubes and then incubate at water bath of 37 degree, optimal temp for endonuclease to recognize and cut DNA for 30 mins
   4. Add a coloured dye to the mix and inject into the wells of agarose
      
      5. Turn on electricity and watch it move for 5 mins

Question 25

What happens in the DNS REDOX reaction

Answer 25

when added to the boiling water, maltose reduces the DNS turning it a orange colour proportionate to the amount of maltose present - as a result maltose is oxidized into maltonic acid

Question 26

Formula for figuring out Final Concentration of a Solution

Answer 26

C2 = C1-V1/V2 C1: initial concentration of solution V1: initial volume of solution V2: final volume of solution

Question 27

Where can Restriction Endonucleases be found

Answer 27

Only in prokaryotic bacterial cells

Question 28

Bioinformatics:

Answer 28

the use of computer software and computational tools, for the analysis of protein and nucleic acid sequence information

Question 29

Significant of Homology in sequence alignments of proteins or nucleic acids

Answer 29

helps compare identities and similarities and (ii) determine evolutionary relationship - It can also be used when a novel protein is discovered and information is needed about its function

Question 30

Alpha Helices

Answer 30

- will form when there is a hydrogen bond formation between every 4th amino acid, causing the chain to spiral. - alpha helices provide flexibility

Question 31

Thermostable:

Answer 31

being less destroyed or affected by heat - the more thermostable a protein is, the higher percentage of beta sheets it has and the less percentage of alpha helices

Question 32

Fold Family:

Answer 32

contains proteins that have the same major secondary structures in the same arrangement with the same topological connections and are clearly related by evolution. - Folds are formed because of thermodynamic stability

Question 33

Structural Domain

Answer 33

physically independent regions of the tertiary structure, which have a specific function

Question 34

Conserved Def for Boxshade alignment

Answer 34

the same amino acid is found at the same location in different organisms same enzyme

Question 35

Beta Pleated Sheet

Answer 35

- A beta sheet will form when there is a hydrogen bond attaching two parallel polypeptide chains together. - Beta sheets provide rigidity

Question 36

Which DNA primer sequences have higher annealing temperatures

Answer 36

because they have higher concentrations of C-G bp

Question 37

number of chromosomes at different stages if normal human cell has 46 chromosomes

Answer 37

G1: 46 unreplicated (1 chromatid) S: 46 replicated(two sister chromatids) G2: 46 replicated Prophase: 46 replicated Metaphase: 46 replicated Anaphase: 92 seperate chromatids, 46 replicated Telophase/Cytokinesis: 2 cells each with 46 replicated

Question 38

why did we use onion cells and white fish blastula cells for mitosis prac

Answer 38

because the cells are constantly dividing allowing us to see all stages of mitosis clearly