Relaxed plasmids & Regulation of copy number

1. &
Regulation of copy number
RELAXEDPLASMIDS
Maryam Amini
M.Sc student of Biotechnology
Kerman Graduate University Of Technology

2. Based upon the number of copies per cell, plasmids are
classified into two types.
1. Stringent plasmids
These plasmids exist in small numbers, i.e. <100 copies/cell.
Stringent plasmid is under the control of bacterial genome for
replication and segregation. Generally, conjungtive plasmids
are mostly stringent plasmids
2. Relaxed plasmids
These plasmids exist in large numbers, i.e., > 100 copies/cell.
Relaxed plasmid is not under the control of bacterial genome
for replication and segregation. Generally, relaxed plasmids are
of low molecular weight and most of them are of the non
conjugative type.
CLASSIFICATION OF
PLASMIDS

3.  The ability of certain plasmids to continue to replicate after their
bacteria stop dividing, the result is a single bacteria that contains
hundreds of plasmids.
 A plasmid that replicates independently of the main bacterial
chromosome and is present in 10-500 copies per cell
 The plasmids used in molecular cloning, however, are under
relaxed control; they are normally present in 10 to as many as
700 copies per cell.
 Moreover, protein synthesis in the bacterial host is inhibited, for
example, by the antibiotic chloramphenicol, thereby preventing
cell division. These plasmids continue to replicate until two or
three thousand copies have accumulated per cell.
 The plasmids that have been constructed for molecular cloning
are relatively small, replicate under relaxed control, carry genes
specifying resistance to one or more antibiotics, and contain a
number of conveniently located restriction endonuclease sites
into which the DNA to be cloned may be inserted.

4. Stringent and Relaxed Plasmids - Plasmid SC101, and its derivatives
are called stringent plasmids, while Co1E1 and its derivatives are
relaxed plasmids with respect to DNA replication the differences
between the two are as follows:
pSC101 (Stringent) Co1E1 (relaxed)
1.Exponentially growing cells have 6-
8 plasmids per choromosome.
Exponentially growing cellls have 20-
30 plasmids.
2.Plasmid replication requires new
protein synthesis. No new protein synthesis required.
3.Plasmid replication does not require
an active DNA polymerase I.
Active DNA polymerase I required.

5. REGULATION OF COPY
NUMBER
ColE1 plasmids:
• Many of the most popular lab plasmids were derived from ColE1 or
pMB1, which is very similar; eg. pBR322, pUC series,pBAD series,
pACYC184 and the pET series.
• ColE1 plasmids use an antisense RNA called RNA I that interferes
with processing of RNA II to the form that serves a primer to initiate
replication by Pol I.
• The more copies of the plasmid, the higher the concentration of RNA
I to inhibit replication.
• RNA I and RNA II are transcribed from the same region of DNA but in
opposite directions using different, complementary template strands.
• Binding of RNA I to RNA II prevents it from binding to OriV being
processed to its primer form by RNase H.
• The protein Rop stabilizes the interaction between RNAs I and II.
• Note that no plasmid-encoded protein is necessary for replication.

8. Unilike ColEI, most plasmids require a plasmid encoded protein,
called Rep, that separates the two strands of DNA at OriV to initiate
replication.
R1 plamids:
• R1 plasmids use an antisense RNA to inhibit synthesis of Rep
protein, which is necessary for replication.
• The antisense RNA binds to and inhibits translation of an upstream
orf to which the Rep gene is translationally coupled.
• The concentration of antisense RNA in the cell reflects the plasimd
copy number. If the number of plasmids falls below the copy
number, the lower antisense RNA concentration allows Rep to be
translated and replication results.

10. • plasmids such as RK2, F and R6K use a protein called
RepA, which is necessary for replication, to regulate copy
number in two ways.
• First, RepA negatively autoregulates its own transcription.
The more copies of a plasmid in the cell, the more RepA
transcription in repressed.
• Second, when a plasmid is in multicopy, RepA binds two
of them together, which prevents replication instead of
initiating it, as is the case when only one plasmid is bound.
• This method is used by some stringent plasmids.

12. References:
1. http://www.molecular-plant-biotechnology.info/bacterial-vectors/classification-of-plasmids.htm
2. www.mondofacto.com/facts/dictionary?relaxed+plasmid
3. http://www.thefreedictionary.com/relaxed+plasmid
4. Principles of gene manipulation and genomics/ By S. B. Primrose, Richard M.
Twyman/chapter4/basic biology of plasmid and phage vector/64-68
5. Voet, D. and Voet, J. G. Biochemistry John Wiley and Sons,Inc. New York,
1995.
6. Sarah Jandle and Richard Meyer,2006,Stringent and Relaxed Recognition of oriT by Related
Systems for Plasmid Mobilization: Implications for Horizontal Gene Transfer, JOURNAL OF
BACTERIOLOGY, 499–506 Vol. 188, No. 2, American Society for Microbiology.