Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cell’s chromosomal DNA. These extrachromosomal DNAs exist in a parasitic or symbiotic relationship with their host cell and like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division.
Plasmids most commonly used in recombinant DNA technology are those that are engineered to optimize their use as vectors in DNA cloning have three common region essential for DNA cloning (Figure 2.6): a replication origin; a marker that permits selection, usually a drug resistance gene; and a region in which exogenous DNA fragments can be inserted (Lodish, 2004).
Figure 2.6 Basic components of a plasmid cloning vector that can replicate within an E.
coli cell (Lodish, 2004)
22 pUC series vectors are primarily designed for general cloning and sequencing. They can be used as a sub-cloning vector or can also be used for expression purposes.
In previous studies, the gene encoding benzaldehyde lyase enzyme was purified and cloned to pUC18 cloning vector (Hinrichsen et. al., 1994) and the enantioselective synthesis ability of BAL and the bioprocess operation parameters were investigated with various Escherichia coli strain using recombinant pUC18::bal plasmid (Demir et al., 2001, 2002, 2003 and Çalık et.
al., 2004, 2006). The bal gene in this modified pUC18::bal plasmid was under the control of hybrid trc promoter (Figure 2.7) and expressed in different E. coli strains. In this study, the pUC19 cloning vector (Figure 2.8) was used for application of sub-cloning for extracellular secretion of the enzyme.
A series of expression vectors are available that are designed to reach the high-level expression of the foreign gene in E. coli. One of these genetically engineered expression vector is pRSETA (Figure 2.9). This plasmid has several useful features as a vector such as having T7 promoter to control the expression of the gene of interest; ribosome binding site which has optimum space from the multiple cloning site for efficient translation; multiple cloning site containing 11 restriction enzyme recognition sequence; T7 terminator permitting efficient transcription termination; f1 origin to allow single strand rescue; ampicillin resistant gene; and pUC origin to provide high copy replication and growth in E. coli (http://www.invitrogen.com).
For the gene cloning in Bacillus species, among the constructed vectors, E. coli/B. subtilis shuttle vector pRB374 was selected (Figure 2.10). In this designed vector, eleven unique restriction sites flanked by two transcriptional terminators, ampicillin resistant gene for selection in E. coli and kanamycin resistant gene for selection in B. subtilis are available. The genes cloned into pRB374 from the multiple cloning site are under the control of B. subtilis vegII promoter which can initiate transcription in both B. subtilis and E. coli (Brückner et. al., 1992).
23 ori amp
Ptrc
pBAL6308 bp
BglII
HindIII BAL
His-Tag
Figure 2.7 Modified pUC18::bal plasmid (Pohl et. al., 2002)
Figure 2.8 pUC18/19 cloning vector (http://www.fermentas.com)
24 Figure 2.9 pRSET A,B,C expression vectors (http://www.ivitrogen.com).
neo bla
pRB374 5.8 bp T0
T0 T1
Bgl I
ble T1 p
Bgl II EcorI SacI KpnI SmaI BamHI XbaI
SaII AccI PstI SphI HindIII
Figure 2.10 pRB374 expression vector (Brückner et. al., 1992).
25 2.3.7 Ligation Reaction and Transformation
The DNA fragment of interest cut by a restriction enzyme leads to single stranded tails, sticky ends, which have a tendency to anneal with the complementary strand present in the reaction mixture. The addition of vector DNA cut open by the same restriction enzyme results in the annealing of the foreign DNA to the complementary ends of the cut vector. The phophodiester bonds missing between the attached strands (Figure 2.11- indicated by arrows) is covalently bond by DNA ligase. This enzyme catalyzes the condensation of 3’-hyroxyl group with a 5’-phosphate group to add the missing links.
The ligation reaction is the rate limiting step in genetic engineering techniques since this reaction requires the cohesive ends of foreign DNA and open plasmid DNA to attach in correct orientation and anneal while preventing the relegation of opened vector DNA. Therefore, optimum ligation reaction conditions should be determined by both paying attention to foreign DNA and plasmid DNA concentrations. Reclosure of the vector can be minimized by treating the opened vector with phosphatase (Çalık et. al., 1998; Bailey, 1986 and Glazer, 1995).
After ligation, the mixture bearing the desired vector-donor combinations is then moved into the recipient or host cell. In most cases this is done by transformation (Schuler and Kargı, 2002).
There are four different methods for direct introduction by transformation:
1. Natural transformation: Foreign DNA is taken up by the bacteria and fused to the chromosomal DNA of the organism. The genetic and physiology of naturally transformable species are not well know, however, with the exceptions of Bacillus subtilis.
2. Artificial transformation: The most preferable method for E. coli cells is to convert the cells into a competent state by resuspension in buffer solutions containing very high concentrations of CaCl2 at 0oC. The effect of Ca+2 on a membrane bilayer with a high content of acidic lipids is to freeze the hydrocarbon interior, presumably by binding tightly to the negatively charged head groups of lipids.
26 Because the outer membrane of Gram-negative bacteria such as E.
coli contains a large numbers of acidic groups at a very high density, this membrane becomes frozen and brittle, with cracks through which macromolecules, including DNA, can pass. After DNA is added to the suspension, the cells are heated to 42oC and then chilled. Under these conditions, cells have been found to take a pieces of DNA through the cytoplasmic membrane.
3. Protoplast transformation: Enzymes are used to hydrolyze the rigid cell wall to convert the cell into protoplast bounded by the cytoplasmic membrane.
4. Electroporation: Applying short electrical pulses of very high voltage is believed to reorient asymmetric membrane components that carry charged groups, thus creating transient holes in the membrane. DNA fragments can then enter through these openings, presumably by spontaneous diffusion (Glazer, 1995).
Figure 2.11 Mechanism of ligation reaction (http://www.biologymad.com)
27 2.3.8 Selection and Screening of Recombinant Plasmids
After transformation, it is important to note that construction of the desired vector-donor DNA usually results in a mixture including some opened or rejoined (without donor DNA) vector molecules, or insertion of DNA contaminants of donor DNA into the vector. Consequently, an efficient method to screen transformants for those with the desired vector-donor DNA combination is important (Schuler and Kargı, 2002).
Antibiotic resistance is most commonly used selection method because it allows for an extended host range and provides more flexibility in growth conditions (Smith, 1995). After transformation, cells possibly carrying the recombinant plasmid with the antibiotic resistance gene, are spread on plates containing a particular antibiotic to select the antibiotic resistant cells. Not all of cells growing on the plates with specific antibiotic carry the desired vector-donor DNA combination. Candidates are further grown and the plasmids they carry are isolated and cut with proper the restriction enzymes to check whether they carry the correct combination.
Insertion of a foreign gene piece of DNA into the vector could be detected by the inability of the cells containing the plasmid with the gene encoding the enzyme β-galactosidase, which can be screened on plates by a color assay.
Restriction enzyme recognition sequences in the polylinker region is situated within the fragment of the E. coli LacZ gene (lacZ’). When the E. coli are grown on agar plates containing a colorless compound called X-GAL, cleavage of the X-GAL by the enzyme produces an insoluble blue product. Thus, these E. coli colonies with plasmids that do not have an insert of foreign DNA are blue while plasmids with an insertion of foreign DNA yields white colonies because of the shifts in the lacZ reading frame which causes β-galactosidase not to be produced (Watson, 1992). By this way, new recombinant cells can be selected.
This process is called as blue-white screening.
The expression plasmid, pRB374, carries both kanamycin and ampicillin resistance gene for selection of correct transformants in Bacillus species and E.
coli strains, respectively, from the culture.
28 Both the cloning vector, pUC19, and expression vector, pRSETA, provide ampicillin resistance for selection. The multiple cloning region of pUC19 vector lies on the lacZ gene fragment for screening the recombinants by blue/white colony screening.