The replicons of plasmids are generally different from the those used to replicate the host's chromosomal DNA, but they still rely on the host machinery to make additional copies. ORI sequences are generally high in As and Ts. Why, you ask? Well, A-T base pairs are held together with two hydrogen bonds not three as G-C pairs are. As a result, stretches of DNA that are rich in A-T pairs melt more readily at lower temperatures. When DNA melts, it gives the replication machinery room to come in and get busy making copies.
So Many Origins, So Little Time
There are lots of ORIs out there so, for simplicity’s sake, we've ignored those used in eukaryotic cells and viruses and focused only on those found in bacteria. Some common ones you might see include ColE1, pMB1 (which comes in a few slightly different but well known derivatives), pSC101, R6K, and 15A. Not all origins of replication are created equal. Some will produce many plasmid copies and others produce just a few copies depending on how they are regulated. Generally, control of replication is referred to as "relaxed" or "stringent" depending on whether the ORI is positively regulated by RNA or proteins, respectively. A plasmid's copy number has to do with the balance between positive and negative regulation and can be manipulated with mutations in the replicon. For example, the pMB1 ORI maintains about 20 copies per cell, while pUC – which differs by only two mutations – will produce as many as 700 copies per cell.So, how do you choose? Addgene Senior Scientist Marcy Patrick says researchers can ask themselves a few questions to get started: Will the plasmid be used exclusively in E. coli? Gram negative bacteria in general? Both Gram negatives and Gram positives? Will you have only one plasmid type in your cells at a time? Do you want to make a lot of your plasmid? Is the gene toxic in high amounts? It is always good to keep in mind that plasmids with low to medium copy numbers can still express massive amounts of protein given the proper promoter and growth conditions.
Choose Your Origin of Replication Wisely
In other words, the best choice of ORI depends on how many plasmid copies you want to maintain, which host or hosts you intend to use, and whether or not you need to consider your plasmid's compatibility with one or more other plasmids. Generally speaking, plasmids with the same ORIs are incompatible because they will compete for the same machinery, creating an unstable and unpredictable environment. As a rule, plasmids from the same group should not be co-transformed, so if you require two plasmids for an experiment, make sure they have "compatible" ORIs. See the table below for more details.| Common Vectors | Copy Number+ | ORI | Incompatibility Group | Control |
| pUC | ~500-700 | pMB1 (derivative) | A | Relaxed |
| pBR322 | ~15-20 | pMB1 | A | Relaxed |
| pET | ~15-20 | pBR322 | A | Relaxed |
| pGEX | ~15-20 | pBR322 | A | Relaxed |
| pColE1 | ~15-20 | ColE1 | A | Relaxed |
| pR6K | ~15-20 | R6K* | B | Stringent |
| pACYC | ~10 | p15A | B | Relaxed |
| pSC101 | ~5 | pSC101 | C | Stringent |
| pBluescript | ~300-500 | ColE1 (derivative) and F1** | A | Relaxed |
| pGEM | ~300-500 | pUC and F1** | A | Relaxed |
This table defines common cloning
vectors, their copy number, ORI, and incompatibility groups. Note the A
-C compatibility grouping is an arbitrary designation, and plasmids from
the same incompatibility group should not be co-transformed.
+Actual copy number varies. See below for additional considerations.
*Requires pir gene for replication (reference).
**F1 is a phage-derived ORI
that allows for the replication and packaging of ssDNA into phage
particles. Plasmids with phage-derived ORIs are referred to as phagemids.
Other Factors that Affect Copy Number
Although the sequence and regulation of the ORI dramtically affect
the copy number of a plasmid, other external factors contribute as well.
These considerations are especially useful to keep in mind if you are
planning to purify your plasmid DNA:
The insert:
- Bacteria tend to maintain fewer copies of plasmids if they contain large inserts or genes that create a toxic product.
The E. coli strain:
- Most E. coli strains can be used to propogate plasmids, but endA- E. coli are best for high yields of plasmids.
Growth conditions:
- The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect copy number. Some ORIs are temperature sensitive; others ORIs can be "tricked" into amplifying more copies with the addtion of Chloramphenicol – make sure your growth conditions aren't working against you!
The culture inoculum:
- Freshly streaked bacteria have higher copy numbers – for optimal results always pick a single colony and do not subculture directly from glycerol stocks, agar stabs, or liquid cultures.
- Incubation for 12-16 hours tends to give higher copy numbers since the bacteria have just reached stationary phase, but the cells have not started to die off.
Plasmid Replication
In order for a piece of circular, dsDNA to be propagated in bacteria, it needs to be replicated by host machinery. There is a sequence in the plasmid that directs the cell to begin replication. Important considerations are host range, compatibly, and copy number. The host range refers to what species of bacteria will recognize the origin of replication and thus allow for replication. The compatibility refers to a plasmid's ability to coexist with another plasmid in the same cell. Copy number refers to the average or expected number of copies of the plasmid per cell.There are three main mechanisms for plasmid replication: Rolling Circle, Strand Displacement, and Theta.
Strand displacement replication
RepC binds repeat sequences recruits RepA ( a helicase) to melt an AT rich region. This exposes two single stranded origins ssiA and ssiB. RepB polymerizes primers for these origins. DNA polymerization follows in each direction, meanwhile displacing the non-template stand.Strand displacement is associated with broad host range vectors, possibly because it does not require any of the normal host machinery (DnaA, DnaB, DnaC, and DnaG)
Rolling circle replication
A nick is made by the Rep protein at the "double strand origin" of a dsDNA plasmdid. The free 3'OH is extended, displacing as it progresses. After one unit length of displacement, replication is terminated, yielding one dsDNA plasmid and ssDNA of one unit length. The displaced strand then serves as a template for replication from a "single strand origins." Since each strand is replicated independently, it is possible for the ssDNA form to accumulate.This mechanism is found in gram-positive bacteria like Staphylococcus aureus and Streptomyces lividans as well as many bacteriophages.
Theta replication
DnaA (often with the help of other proteins) binds the origin at DnaA boxes. This promotes melting of the orgin. This allows DnaC to load to DnaB helicase, opening the origin further. DnaG is then recuited to form a short RNA primer.DNA polymerase III extends this primter. If there is only one leading primer, a single fork circumnavigates the entire plasmid until the origin is reached, and daughter plasmids separate. In bidirectional replication, two forks propagate and meet on the far side of the plasmid before resolution.
Theta is the most common form of DNA replication, including most plasmids as well as chromosomes. It is particularly associated with gram-negative bacteria. ColE1, P15A, RK2, F, and P1 all use theta replication.
Host range
Plasmids are classified as having a narrow or broad host range.- ColE1 and pMB1 are limited to E. coli and a few close relatives,
- RK2 plasmids can be used in most gram-negative bacteria.
- RSF1010 can use used in most gram-negative bacteria, and some gram-positive
- Plasmids from gram-positive bacteria tend to function well in other gram-positive bacteria.
Compatibility groups
If two plasmids have the same (or very similar) origins of replication, they will compete with each other for replication machinery. This results in an unstable situation. If the two plasmids posses different selectable markers, this can be maintained for several generations, but eventually one of the plasmids will be lost. For scenarios in which multiple plasmids are necesary, one must be careful to choose plasmids will compatible origins. The most common dual-plasmid pair is ColE1(or pMB1) and p15A. The most common plasmid triplet is ColE1 (or pMB1),p15A, and pSC101. Tolia and Joshua-Tor suggest the following groups:- ColE1/pMB1 (eg pET, pUC, pBR322, pGEX, pMAL)
- P15A (eg pBad, pACYC)
- CloDF13
- ColA
- RSF1030
Copy number
An important consideration in choosing what plasmid backbone to use is the copy number. For example, cloning is best done with a high copy plasmid (e.g. pUC) as plasmid preps will have a higher yield. Expressing a toxic gene is better from a low to medium copy plasmid(e.g. pET which uses the pBR322 origin), as there are fewer copies.- ColE1: 15-20 copies
- pMB1: 20-700 copies
- pUC: 500-700 copies
- pBR322: ~20 copies
- pSC101: ~5 copies
- P15A: 10-12 copies
- RK2: 4-7 copies
- F1: ~1 copy
- CloDF13: 20-40 copies
- ColA: 20-40 copies
- RSF1030: >100 copies
- P1: ~1 copy
- R6K: 15-30 copies
Control of initiation/copy number
There are several mechanisms by which copy number is controlled. In all cases, some negative-regulating element (RNA or protein) is expressed from the plasmid. As the plasmid concentration increases, so too does the negative regulator. This provides a negative feedback, which stabilizes the copy number. Two plasmids that are regulated by each other's regulator will not be compatible.RNA regulation
ColE1/pMB1: The origin contains regions promoting the synthesis of RNA I and RNA II. RNA II hybrizes to the DNA, yielded a DNA/RNA hybrid which can serve as a substrate for RNaseH. Digestion of RNA II by RNaseH yields the primer for replication. RNA I binds and sequesters RNA II, so it is unavailable for RNAse H digestion. As the copy number increases, so does the concentration of RNA I. This provides a negative feedback for replication, and sets the average number of plasmids per cell.Additionally, The Rop protein helps lower the copy number, by stabilizing the RNA I/ RNA II duplex. Deletion of Rop, as well as a point mutation that weakens the RNA I and RNA II duplex, accounts for the higher copy of pUC (a pMB1 derivatives)
P15A, ColA, RSF1030, and CloDF13 are similar, but with versions of RNA I and RNA II that sufficiently different to allow for compatibility.
RNA and protein regulation
On the R1 plasmid, OriR is bound by RepA, thus promoting replication by recruiting DnaA. RepA can be expressed from two different promoter. A proximal promoter (pRepA) drives only RepA while a distal promoter (pCopB) drives both CopB and RepA. CopB represses pRepA, thus once there are enough plasmids around, CopB levels become high enough to limit RepA expression to pCopB promoter. plasmid encoded CopA is completmentary, and thus binds to the 5' end of the transcript originating from the pCopB promoter. The dsRNA is a substrate for the processive RNase III.Iteron regulation
Like the above examples, pSC101's replication is positively regulated by RepA binding the origin. RepA is also used to control copy number, by two mechanisms.Firstly, RepA negatively regulates its own transcription, thus the RepA protein levels (and its ability to promote replication) is confined to narrow limits.
Secondly, The plasmid contains several (3-7) repeats of a 17-22bp sequence called iteron sequences. RepA binds the iterons, and at higher plasmid conncentration, this can lead to "handcuffing" of two plasmids. Interestingly, adding extra iteron sequences on other plasmids can reduce the copy number by this handcuffing mechanism.
F, RK6, P1, RK2, and RP4 also use iterons, but the regulating protein and origins differ.
pETcoco is an interesting plasmid, made by Novagen. It can be maintained as a single copy plasmid using the origin and positive regulator from the F plasmid (oriS and RepE). It can be swiched to a medium copy plasmid using the machinery from the RK2 plasmid (oviV and trfA). The switch is achieved by the induction of the trfA protein, which binds and iteron on oriV, thus promoting initiation from this origin by aiding in melting and recruitment of DnaB.
