Workshop I

Question 1

What are plasmids?

Answer 1

Extrachromosomal replicons found in Arachea, Bacteria and Eukaria.

Question 2

What is the ternary structure of the plasmid in native conditions and what can influence it?

Answer 2

In the bacterial cytoplasm, plasmids exhibit supercoiled structure, which can be influenced by topoisomerases. supercoiled –topoisomerase I–> relaxed
relaxed –topoisomerase II + ATP–> supercoiled

Question 3

What are topoisomers?

Answer 3

DNA fragments differing in their “topography”, not sequence.

Question 4

What are two types of plasmids regarding the copy number and what are their features?

Answer 4

Relaxed plasmids - high copy number, often used in cloning
Stringent plasmids - low copy number (1 to few per cell), replicate only with bacterial chromosome, par genes are responsible for sorting them to both daughter cells to prevent the loss of plasmid.

Question 5

What is plasmid incompatibility?

Answer 5

Plasmid incompatibility is the inability of 2 plasmids to coexist in 1 bacterium due to the similarity of their replication or segregation mechanism. Plasmids from 1 incompatibility group are compatible with plasmids from different incompatibility group.

Question 6

How can the plasmid number be controlled?

Answer 6

A rop protein dimer can initiate binding of the replication products from 2 forks of replication, thus inhibiting the entire process.

Question 7

What are the features of plasmids used in gene technology and why are these features necessary?

Answer 7

• convey at least 1 marker gene that can be used for artificial selection and a polylinker region within that gene. - to distinguish between recombinant plasmids and the non-recombinant.
• neither transmittable (via conjugation) nor mobilizable (made transmissible with a helper plasmid) - biological safety
• small molecular weight - for increased efficiency of transformation
• copy number under relaxed control –> high copy number –> more successfully cloned inserts.

Question 8

How Ethidium Bromide can be used to modulate plasmids?

Answer 8

It can alter the ternary structure of the plasmids giving them more positive twist.

Question 9

What are the pUC19 features?

Answer 9

1) length - 2.7 kbp
2) Ori - mutated pMBI
3) rop gene - deleted
- therefore high copy number (500-700/cell)
4) resistances - bla (Tn3) gene (beta lactamase), tet deletion ( tetracycline)
5) MCS introduced, restriction sites at other locations removed
6) lacZ - regulatory seq and only lacZ gene - allows for blue/white screening

Question 10

What are the pBR322 features?

Answer 10

1) length - 4.4 kbp
2) Ori - pMBI
3) rop gene - low copy number (15-20/cell)
4) resistances - bla gene & tet gene - identification through insertional inactivation

Question 11

How much units of enzyme do we need per 1 ug og DNA?

Answer 11

=(length of sample DNA* #of cut sites in vector)/(length of vector*#cut sites in sample)