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NAME: Loshieni Shri Gunasegaran

NPM: 260110152020

Quiz Modul 4. DNA Plasmid Isolation

Deadline: Sunday, 18 March 2018 10.00pm GMT+7

1. Why we use E.coli as a host?


2. Mention 3 purposes of plasmid?
3. Why plasmids can be used as cloning vectors?
4. What si RNase A? Explain its purpose!
5. Lysis buffer contains NaOH and SDS detergent, explain what does NaOH do in the lysis
buffer!
6. When bacterial cells are grown in broth culture, what is used to make sure that only
bacteria that contain the plasmid will grow? Explain briefly
7. What does SDS do in lysis buffer?
8. Explain briefly 3 stages of DNA plasmid isolation

Answers

1. a) Genetically Simple structured .


They have small genome size compared to eukaryotes.E. coli, live their entire lifetime in a
haploid state (having a single set of unpaired chromosomes).So there is no second set of
chromosomes to mask the effects of mutations during protein engineering experiments.
b) Growth Rate

E. coli grows rapidly at a rate of one generation per twenty minutes under typical growth
conditions.Allows genetic experimental results in mere hours instead of several days, months, or
years.Faster growth also means better production rates when cultures are used in scaled-up
fermentation processes.

c) Safety
E. coli is naturally found in the intestinal tracts of humans and animals where it helps provide
nutrients (vitamins K and B12) to its host. E. coli are generally relatively innocuous if handled
with reasonable hygiene.( David Summers ,1996 )

2 Functions of plasmid

- Degrading or digesting dead organic matter


- Produce antibiotics which is involved in killing other strains of bacteria
- Involved in bacterial conjugation (David P. Clark, 2012).

3. A plasmid can be moved from cell to cell easily. They also contain the genes that control
their own replication. Therefore they can be used to move and to copy any gene that can be
inserted into the plasmid. Plasmids also do not usually have detrimental effects on their host
cells. (Andrew Preston, 2003)

4.RNASE A is added to remove the DNA from RNA.It is done to obtain pure DNA without the
contamination of RNA. (Thomas, 2008)

5. The lysis buffer contains sodium hydroxide (NaOH) and the detergent Sodium Dodecyl
(lauryl) Sulfate (SDS). SDS is there to solubilize the cell membrane. NaOH helps to break
down the cell wall, but more importantly it disrupts the hydrogen bonding between the DNA
bases, converting the double-stranded DNA (dsDNA) in the cell, including the genomic DNA
(gDNA) and your plasmid, to single stranded DNA (ssDNA). This process is called
denaturation and is central part of the procedure, which is why it’s called alkaline lysis. SDS
also denatures most of the proteins in the cells, which helps with the separation of the proteins
from the plasmid later in the process. (Schumann ,2008.)

6. After transformation, bacteria are grown on a nutrient rich food called agar. Only bacteria
containing a plasmid with antibiotic resistance will grow in the presence of antibiotic. Antibiotics
were added into the broth medium. This is to ensure that only bacteria that contain the plasmid
will grow. This is because of plasmids can carry one or more antibiotic resistance genes, which
confer resistance to a specific antibiotic to the bacteria carrying them. The presence of an antibiotic
resistance gene on a plasmids allows researchers to easily isolate bacteria containing that plasmid
from bacteria that do not (Lederberg J ,1952)
contain it by artificial selection

7. Most lysis buffers contain salts (e.g. Tris-HCl or EDTA) to regulate the acidity and
osmolarity of the lysate.Detergents (such as Triton X-100 or SDS) are added to break up
membrane structures. Detergents are organic amphipathic (with hydrophobic tail and a
hydrophilic head) surfactants. They are used to separate membrane proteins from membrane
because the hydrophobic part of detergent can surround biological membranes and thus isolate
membrane proteins from membranes.Detergents are often categorized as nonionic, anionic,
cationic, or zwitterionic, based on their hydrophilic head group feature.SDS is an ionic
detergents will disrupt protein functions. Detergents are the primary ingredient that determines
the strength of a buffer.( Lederberg J ,1952)

8. Three stages of DNA plasmid isolation

(Gerdes K,1986)

Reference

1. David Summers ,1996. "Chapter 1 - The Function and Organization of Plasmids". The
Biology of Plasmids. Wiley-Blackwell; First Edition..

2. David P. Clark; Nanette Jean Pazdernik, 2012. Molecular Biology (2nd ed.). Academic
Cell. p. 795.
3. Andrew Preston, 2003, "Chapter 2 - Choosing a Cloning Vector". In Nicola Casali,
Andrew Preston. E. Coli Plasmid Vectors: Methods and Applications. Methods in
Molecular Biology, Vol. 235. Humana Press.

4. Kandavelou K, Chandrasegaran S ,2008. "Plasmids for Gene Therapy". Plasmids:


Current Research and Future Trends. Caister Academic Press.

5. Thomas, Christopher M; Summers, David, 2008. "Bacterial Plasmids". Encyclopedia of


Life Sciences.

6. Schumann ,2008. "Chapter 1 - Escherichia coli Cloning and Expression Vectors". In


Georg Lipps. Plasmids: Current Research and Future Trends. Caister Academic Press. pp.

7. Lederberg J ,1952. "Cell genetics and hereditary symbiosis". Physiol. Rev. 32 (4): 403–
430. PMID 13003535.

8. Gerdes K, Rasmussen PB, Molin S ,1986. "Unique type of plasmid maintenance


function: postsegregational killing of plasmid-free cells". Proc. Natl. Acad. Sci. U.S.A.