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I'm looking for suggestions as my internet searches aren't turning up much.
I'm trying to find a plasmid that has the pBR322 origin, as it's low(ish) copy number and can tolerate large inserts. Additionally I need the plasmid to be inducible with a system other than the pBAD setup (e.g. IPTG would be fine, but if there is one that is more tightly repressed that would be preferrable as IPTG is leaky).
Lastly, I could do with it having a potentially obscure resistance cassette, but this is less of a problem.
Any help appreciated.
The pET vectors family is what you are looking for.
Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli
The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA+ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA+ gene was expressed in the presence of the inducer isopropyl-1-thio-beta-D-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30 degrees C, or after inactivation of DnaA by a shift to 42 degrees C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom- plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately.(ABSTRACT TRUNCATED AT 250 WORDS)
PET Bacterial Recombinant Protein Vector
The pET vector system is a powerful and widely used system for expressing recombinant proteins in E. coli. The gene of interest is cloned into the pET vector under the control of the strong bacteriophage T7 transcription and translation regulatory system. Activation of expression is achieved by providing T7 RNA polymerase within the cell. When the system is fully induced, nearly all of the cell&rsquos resources are devoted to expressing the gene of interest. With just a few hours of induction, the recombinant protein could comprise nearly half of the cell&rsquos total protein.
For further information about this vector system, please refer to the papers below.
|Methods Enzymol. 185:60-89 (1990)||Use of T7 RNA polymerase to direct expression of cloned genes.|
|J Mol Biol. 219:45 (1991)||Development of the T7lac promoter system.|
The gene of interest is initially cloned into the pET vector in a bacteria host that lacks the T7 RNA polymerase gene. This eliminates plasmid instability due to expression of proteins of interest that may be harmful to host cells. Afterwards, expression of the gene of interest can be initiated in two possible ways. The host cells can be infected with phage carrying the T7 RNA polymerase gene (e.g. &lambdaCE6 phage), or more commonly, the pET plasmid can be transferred into a bacteria host strain whose genome has been engineered to carry the T7 RNA polymerase gene under the control of theLacUV5 promoter. Expression of the T7 polymerase is induced by the addition of the lactose analog IPTG to the bacterial culture.
The pET vector exists as a low copy number plasmid in host E. coli, which reduces leaky expression before induction. The vector utilizes the T7lac promoter system for strong and tightly controlled gene expression. In this system, there is a T7 promoter that can be acted upon by T7 RNA polymerase to drive high-level expression of the gene of interest. Additionally, there is a lac operator (LacO) sequence just downstream of the T7 promoter that can be acted upon by the lac repressor (LacI) protein to block transcription of the T7 promoter. The plasmid also carries the natural promoter and coding sequence for LacI. The LacI protein acts at the LacUV5 promoter in the host cells to repress expression of the T7 RNA polymerase gene by the host polymerase, and also functions at the T7lac promoter on the pET vector to block transcription of the gene of interest by any T7 RNA polymerase that may be made due to leaky expression. Addition of IPTG blocks the inhibitory action of LacI, thereby inducing expression of T7 RNA polymerase and also removing LacI inhibition of the gene of interest.
Although the pET expression system is designed for high-level recombinant protein expression, the expression level can be reduced by decreasing the amount of IPTG supplied to host cells. This can be advantageous when expressing proteins with limited solubility. Additionally, the system is able to maintain the gene of interest in a transcriptionally silent state when T7 RNA polymerase is not present.
All custom pET vectors will be supplied in an E. coli strain designed to maximize plasmid integrity and lacking the T7 RNA polymerase gene (such as Stbl3). For recombinant protein production, we recommend transferring the vector to BL21(DE3) or HMS174(DE3) host bacteria strains, which carry chromosomal copies of the T7 RNA polymerase gene driven by the LacUV5 promoter. In cases when toxicity of the gene of interest is an issue in these expression host strains, the use of hosts carrying the pLysS or pLysE plasmids may be beneficial. These plasmids suppress basal expression of the gene of interest by producing T7 lysozyme, a T7 RNA polymerase inhibitor.
Strong expression: The T7 transcription and translation regulatory system allows for very high-level production of proteins of interest, in many cases close to 50% of total protein in the culture.
Tightly controlled expression: The expression of the gene of interest is generally very strongly repressed in the absence of added IPTG, and this &ldquooff&rdquo state is very robust for most genes of interest in most host strains.
Host requirements: Completed pET vectors should be maintained in an E. coli strain lacking the T7 RNA polymerase gene, and must be transferred to a separate host strain containing the T7 RNA polymerase gene before induction of protein expression.
Potential leaky expression in some hosts: Even in the absence of IPTG, there may be some low-level expression of T7 RNA polymerase from the LacUV5 promoter in some expression host strains, which could lead to bacterial toxicity for certain genes of interest in certain host strains.
T7 promoter: Drives high-level transcription of the gene of interest when T7 RNA polymerase is present. When placed immediately upstream of a LacO element, the entire cassette is known as the T7lac promoter.
LacO: Binding site for LacI. This element inhibits activity of the T7 promoter when LacI protein is present, preventing leaky expression of the gene of interest.
RBS: The ribosome-binding site and translation initiation element from T7 bacteriophage. This allows for efficient production of the protein of interest.
ORF: The open reading frame of your gene of interest is placed here.
T7 terminator: Signal sequence to terminate the transcript made from the gene of interest, preventing run-on transcription.
Ampicillin: Ampicillin resistance gene. It allows the plasmid to be maintained by ampicillin selection in E. coli.
pBR322 ori: pBR322 origin of replication. Plasmids carrying this origin as well as the Rop gene exist in low copy numbers in E. coli.
Rop: Repressor of primer. It encodes a small protein that regulates plasmid copy number. The presence of the Rop protein, in combination of pBR322 origin of replication on the plasmid, results in low copy numbers of the plasmid.
Plant Gene Expression Agrobacterium Binary Vector
Agrobacterium-mediated genetic transformation using binary vectors is a powerful and effective method for generating transgenic plants. This system utilized the ability of the bacteria Agrobacterium tumefaciens to insert foreign DNA into the genome of cells of numerous plant species.
The Agrobacterium binary vector system is derived from natural tumor-inducing (Ti) plasmids. Agrobacterium transfers a region of the Ti-plasmid known as the transfer DNA (T-DNA) into numerous plant species, where it is integrated into the host genome. The T-DNA is delineated by flanking 25 bp T-DNA border repeat sequences, in direct orientation with one another. In our binary vector system, all tumor-associated intervening T-DNA sequences have been removed, leaving the T-DNA border repeats, which flank and direct host integration of the user&rsquos sequence of interest.
The complete binary vector system consists of two parts. The first, referred to as the T-DNA binary vector (or simply &lsquobinary vector&rsquo), contains two T-DNA repeats bracketing the DNA sequence which will be inserted into the plant host. The user&rsquos gene of interest is cloned into this portion of the binary vector. The second plasmid, referred to as the vir helper plasmid, encodes components necessary for integration of the region flanked by the T-DNA repeats into the genome of plant cells. Prior to transforming the target plant cells, these two plasmids are brought together in Agrobacterium tumefaciens by co-transformation, co-electroporation, or conjugation.
When the binary vector and the vir helper plasmid are both present in the same Agrobacterium cell, proteins encoded by the vir helper plasmid act in trans on the T-DNA border repeat elements to mediate processing, secretion, and host genome integration of the sequence between the left and right border repeat elements. Insertion occurs without any significant bias with respect to insertion site sequence.
For further information about this vector system, please refer to the papers below.
|Plant Physiology. 146:325-32 (2008)||Review of T-DNA binary vector systems.|
|Trends in Plant Sci. 5:446-51 (2000)||Review of T-DNA binary vector systems.|
Our Agrobacterium binary vector system enables efficient insertion of sequences into genomes of target plant cells. The binary vector plasmid is optimized for replication in E. coli and Agrobacterium, efficient integration of user selected sequences into target plant cells, and high-level expression of transgenes.
Permanent integration of vector DNA: Conventional transfection results in almost entirely transient delivery of DNA into host cells due to the loss of episomal DNA over time. This problem is especially prominent in rapidly dividing cells. In contrast, transformation of plant cells with Agrobacterium vectors can deliver genes permanently into host plant cells due to the integration of the T-DNA region into the host genome.
Technical simplicity: Transformation of Agrobacterium with binary vectors is technically straightforward, as is transformation of plant cells using binary vectors and Agrobacterium.
Very large cargo space: Our Agrobacterium binary vector system can accommodate very large DNA inserts. Generally, inserts up to 20kb can be efficiently cloned transformed into target cells.
3&rsquo deletions: Within the plant, it is common for nucleolytic degradation to delete sequence from the T-DNA left boundary (e.g. 3&rsquo) end. However, this is generally not a significant concern since the user&rsquos sequence of interest is cloned near the right boundary, so degradation from the left boundary loss affects the marker gene.
Matching Agrobacterium strains and markers: Care must be taken to select Agrobacterium strains that work effectively with the specific binary vector and markers being used. For example, some Agrobacterium strains already express resistance to certain antibiotics, and binary vectors with these selection markers cannot be used in these strains.
Integration of backbone sequences: In some cases, integration of vector backbone sequences may occur along with T-DNA boundary-flanked sequence. This phenomenon occurs less frequently when low copy Agrobacterium plasmids are used, such as in our binary vector system.
Promoter: The promoter driving your gene of interest is placed here.
Kozak: Kozak consensus sequence. This is placed in front of the start codon of the ORF of interest to facilitate translation initiation in eukaryotes.
ORF: The open reading frame of your gene of interest is placed here.
Nos pA: The nopaline synthase polyadenylation signal of Agrobacterium tumefaciens. This facilitates transcription termination of the upstream ORF.
RB T-DNA repeat: Right border repeat of T-DNA. Upon recognized by Ti plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred to plant cells.
pVS1 StaA: Stability protein from the plasmid pVS1. Essential for stable plasmid segregation in Agrobacterium.
pVS1 RepA: Replication protein from the plasmid pVS1. Permits replication of low-copy plasmids in Agrobacterium.
pVS1 oriV: Origin of replication from the plasmid pVS1. Permits replication of low-copy plasmids in Agrobacterium.
pBR322 ori: pBR322 origin of replication. Facilitates plasmid replication in E. coli. Plasmids carrying this origin exist in low copy numbers (15-20 per cell) in E. coli if Rop protein is present, or medium copy numbers (100-300 per cell) if Rop protein is absent.
Kanamycin: Kanamycin resistance gene. It allows the plasmid to be maintained by kanamycin selection in bacterial hosts.
LB T-DNA repeat: Left border repeat of T-DNA. Upon recognized by Ti plasmid in Agrobacterium, the region between the T-DNA border repeats is transferred to plant cells.
CaMV 35S_enhanced: A strong chimeric promoter which drives marker expression.
CaMV 35S pA: Cauliflower mosaic virus 35S polyadenylation signal. This facilitates transcription termination and polyadenylation of the marker gene.
Marker: A drug selection gene, allowing selection of plant cells transduced with the vector.
A new revision of the sequence of plasmid pBR322.
A revised sequence in the region immediately upstream from the rop gene of pBR322 is reported. Two base pairs in the accepted sequence do not exist in the plasmid DNA. Specifically, a TA base pair is missing at sequence coordinate 1893 [Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. 43 (1979) 77-90] and an AT base pair is missing at position 1915, giving a total size for pBR322 of 4361 bp. These changes are in a potential translation initiation sequence and probably reflect errors in the original sequence rather than recent evolution of the plasmid.
During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of β-galactosidase activity constitutive in a host containing the I qtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lacα/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lacα peptide by an order of magnitude, allowing selection of recombinants by their Lac − phenotype on MacConkey agar.
Present address: Fisons plc., Pharmaceutical Division, Bakewell Road, Loughborough, Leicestershire, LE11 0RH, England.
Plasmid with ITPG and pBR322 ori - Biology
Required for automous replication of the plasmid using the host's replication machinery.
Almost all commonly used plasmids are based on the ColE1 origin of replication (ori).
It is worth noting that bacterial origins of replication are tightly regulated.
While R factors are smaller than the host genome (10 5 bp compared to 5x10 6 bp), replication of these factors to high copy number in the host places a considerable load on the host replication machinery. Naturally occuring origins of replication are therefore negatively regulated to keep copy number down (typically 5 to 10 copies per cell).
While high copy number is disadvantageous in a natural system, it is a desirable feature in a cloning vector - since the whole idea is to be able to easily isolate substantial quantities of particular DNA sequences. Therefore considerable work has gone into engineering the ColE1 ori such that the negative regualtory mechanisms that limit episome copy number are disabled. Modern plasmid vectors are therefore often called 'runaway replicons' and are present at 100 to 1000 copies per cell.
Selectable markers are essential for the identification of bacteria containing recombinant plasmids. Selection can be divided into two types:
Positive selection is used to identify bacteria that contain a plasmid. The most common markers used for positive selection are the antibiotic resistance genes carried by the original R factors.
While many antibiotics and resistance genes are available, the commonly used ones fall into two general classes:
The ligation of foreign DNA fragments into plasmid vectors is a relatively inefficient process - ligation can either produce recircularized plasmid with no insert, or plasmids containing a foreign DNA insert. These products are then transformed into an antibiotic sensitive bacterial host, an positive selection applied to identify bacteria that contain a plasmid. A second selection system is necessary to distinguish between plasmids that merely recircularized from those that carry a foreign DNA insert.
In order to identify those plasmids carrying a foreign DNA fragment, the site of insertion is chosen such that insertion disrupts a selectable marker - a phenomenon we call insertional inactivation.
Two types of selectable markers are used for negative selection
In order to use antibiotic resistance as a negative as well as a positive selection system, the plasmid vector must carry two different antibiotic resistance genes. An example of such a vector is pBR322.
pBR322 carries both an ampicllin and a tetracycline resistance gene.
The phenotype of bacteria containing the intact plasmid is Amp r Tet r
Insertion of foreign DNA into
the Pst I site located in the Amp r gene results in an Amp s Tet r phenotype.
Conversely, insertion of foreign DNA into the EcoRI, Hind III or Sal I sites located in the Tet r gene results in an
Amp r Tet s phenotype.
Positively selected colonies are then restreaked on positive selective media
(master plate from which we recover our desired vector + insert)
and on the negative selection media.
(Amp or Tet media respectively)
Insertional Inactivation of Enzymatic Activity
While the insertional inactivation of antibiotic resistance works, it requires a lot of manipulation - picking the positively selected bacteria and replating on negative selection media etc. In addition to the tedium of picking colonies, vectors like pBR322 also suffer from a paucity of convenient restriction sites at which to insert the foreign DNA fragemnts.
These limitations promted the development of a set of host-vector systems in which it is possible to positively select for bacteria carrying a plasmid and simultaneously select for insertional inactivation of enzymatic activity. This system is based on our old friend, the
beta-galactosidase gene of the E coli lac operon.
- Plasmids are extra chromosomal, self replicating, usually circular, double stranded DNA molecules found naturally in many bacteria and also in some yeasts.
- Although plasmids are not essential for normal cell growth and division, they often confer useful properties to the host such as resistance to antibiotics that can be selective advantage under certain conditions.
- Size of plasmid varies from 1 to 500 kilo base pairs. No. of plasmid per cell may be 1 to 4 copies or 10 to 100 copies.
- These are several plasmid cloning vectors such as PBR322, pSC102, ColE1, pUC, pRP4, pRK2, pRSF1010, pEY, pWWO, Ti- and Ri-DNA plasmid etc.
PLASMID pBR322: This plasmid contains two different antibiotics resistance genes ampicillin and tetracycline and recognition sites for several restriction enzymes.
PLASMID pUC19: It consists of 2,686 base pairs and possess an amp r gene, lac Z gene and lac I gene.
In pUC19, the multiple cloning sequence (MCS) is incorporated into lac Z gene without interfering the functions of other genes.
- The cell receiving the rDNA is called Host
- Many types of host cells are available E.g. E.coli, yeast, animal or plant cells
- coli is the most widely used organism as its genetic engineering
- DNA is a hydrophilic molecule and it can’t pass through the plasma membrane
- DNA is a hydrophilic molecule, it can’t pass through cell membranes so the bacterial cells must first be made “competent” to take up DNA
- Therefore, certain treatments are made in the host self to become competent to take up rDNA
I) Chemical Method
- Treatment of bacterial cells with specific concentration of divalent calcium. This increase the chances of rDNA entry into the bacterial cell wall through the tiny pores
- Incubation of bacterial cells with recombinant DNA on ice. There after these are placed briefly at 42’C after this again transferred on ice. This process enable the bacteria to take up rDNA
II) Microinjection Method
- In this method, the rDNA solution is directly injected into the nucleus of animal cells
- Capillary class micropipettes or micro injections help to inject the rDNA into host cells
- This is most common in case of animal cells.
III) Gene Guns Method
- This technique is also known as a biolistic technique.
- High velocity particles of Gold or Tungsten coated with rDNA are bombarded on Host Cells
- This method is mostly used in plant cells.
IV) Electroporation method
- Electroporation is a technique used to changing the permeability of cell membrane of cells to uptake rDNA in the medium
- Electroporator instrument used to applying suitable voltage to make permeability of cell
- Electroporation is helpful to transform bacteria, fungi, plant cells and animal cells
High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II
The plasmids pUC18 and pUC19 are pBR322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37 degrees C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30 degrees C. The mutation's effects are enhanced by cell growth at 42 degrees C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.