## TOOL AND TECHNOLOGY

### TOOL AND TECHNOLOGY

1.1.    Recombinant DNA Technology
Every organism in the world carries similar characteristics like their parents. This is because information is passed from generation to generation by genetic material. This can be either DNA or RNA, but mainly DNA acts as the genetic material. Nowadays, it is possible to introduce foreign DNA to any other organism and this technology is known as recombinant DNA technology. It has crossed the kingdom barrier and desired gene can be ligated and amplified in bacterial organism regardless of its source. This lead to the development of another very important field i.e., genetic engineering.
A clone is a population of identical organisms derived from a single parent like the members of a bacterial colony. In the same way, if a foreign DNA is introduced in the plasmid of the bacterial host then the whole bacterial colony harbours the recombinant DNA molecule in the form plasmid. The process of recombinant DNA cloning is shown here
Recombinant DNA Technology steps

ISOLATION OF DESIRE DNA AND PLASMID
Isolation of desire DNA
STEPS
A.     Cell lysis
B.    Precipitation of proteins
C.    Removal of RNA
D.    Precipitation of DNA
Cell lysis
i.    Cell lysis can be carried out using strong denaturants such as Sodium Dodecyl Sulfate.
ii.    Ethylene Diamine Tetra Acetic Acid is added to the cell tysis buffer in order to inhibit the activity of DNases.
iii.    Enzymes such as Proteinase K, Cellulase may be added to the cell lysis buffer in order to permit the break down of cellular components.
Precipitation of Proteins
i.    Proteins can be precipitate using organic solvents such as Phenol Chloroform Isoamyl Alcohol.
ii.    When mixed with an aqueous solution of DNA and proteins, the DNA remains in the aqueous phase and the proteins get trapped in the aqueous, organic interphase.

Removal of RNA
i.    RNA can interfere with the downstream processing of DNA.
ii.    RNA is generally degraded by the use of RNase under acidic conditions.
iii.    The degraded RNA can be removed using a solvent extraction step with chloroform.
Precipitation of DNA
i.    Precipitation of DNA can be carried out using absolute Isopropanol or absolute ethanol.
ii.    These alcohols dehydrate the DNA and it appears as a precipitate.
Cut both DNA by same restriction endonuclease
1.    restriction endonuclease is termed as "Molecular scissors" of molecular genetics.
2.    After isolation both DNA are allowed to be cut by same restriction endonuclease so that both DNA (desire DNA and Plasmid) are cut from the same specific palindromic site in the DNA (the sequence that is same on both antiparallel DNA strand).
3.    It produces both sticky ends (Overhanging ends)

Then Ligase both DNA
Both cut DNA is the mix in a tube so that their sticky ends can match with each other.
DNA ends are Joined by DNA ligase. This DNA called recombinant DNA.
Introduce host DNA
Now recombinant DNA is transferred to host cell by transformation method. For transformation, the host cell is made competent to receive recombinant vector via Cacl2 with 42°C.
Culture on the nutrient agar plate containing antibiotics
Plasmid contains anti the biotic resistant gene. So the host cell which contains this recombinant plasmid will survive in culture having antibiotic. Rest all cells which do not have recombinant plasmid will die.
Isolation of living bacteria grow in culture medium
The living host cell is transferred into nutnutrient-richlture for further growth and can be used for the desired purpose.
Recombinant DNA Technology
The components of Recombinant DNA Technology.
•    Desired gene
•    Vector
•    Enzyme
•    Host cell / Organism
Vector
1.    Plasmid
2.    Phagemid
3.    Cosmid
4.    Artificial chromosome
Plasmid

1.2.    Vectors for gene cloning
Vector is a carrier molecule that allows the transfer of the desired gene to host organism. A DNA molecule should display certain characteristics to act as a vector. Like cloning vector, it should be relatively small (less than 10 kb) in size as large molecules are prone to break and difficult to manipulate. Most important cloning vector should replicate within the host organism so that numerous copies of the desired gene can be obtained. The molecules that can work as vectors are :
1.2.1.    Plasmids
Plasmids are naturally occurring self-replicating, circular, non-essential extrachromosomal DNA molecules. These can be obtained from natural strains of E.coli. Plasmids possess three main properties that make it suitable as a vector. These properties are:-
1.    A replicon: It is also known as the origin of replication. This is prerequisite for replication of plasmid in bacterial host cells.
2.    Selectable marker: It is a gene conferring resistance to an antibiotic. Which helps to select the plasmid containing cells only those cells that possess cloning vector can grow in antibiotic-containing media, commonly used antibiotics are given in the table below

3.    Cloning Site: It is also known as the cleavage site. Cloning site is a sequence enter required of nucleotide which can be identified by the restriction endonuclease.

Cleavage by restriction endonuclease can generate fragments with overlapping (sticky ends) which can be ligated with another fragment. By this way, a recombinant plasmid is constructed. This recombinant plasmid is also known as an artificial plasmid.
Plasmids carry the genes that specify novel metabolic activities which range from catabolism of unusual organic substances to metabolic functions that help the host cells with resistance to antibiotics, heavy metals or viruses (bacteriophages). Therefore antibiotic resistance plasmid containing bacteria can grow normally in the toxic concentration of antibiotics.
The plasmid can be isolated by adding intercalating agent ethidium bromide (EtBr). Addition of Ethidium bromide unwinds the DNA and in excess, it rewinds in opposite direction. This helps in isolation of plasmid DNA.
All plasmids do not exist as circular molecules, some strains of bacteria consist of linear molecules of plasmids for e.g.. Streptomyces species and Borrelia burgdorferi. The ends of these plasmids are prone to digestion by nucleases. The ends of linear plasmids are protected either by forming a hairpin loop by repeated sequences of the end as in Borrelia by covalent attachment of proteins as in Streptomyces.
The plasmids whose function has not been determined are known as cryptic plasmids. Most plasmids possess at least one origin of replication through while it can replicate independently within the bacterial host. Smaller plasmids make use of host cell’s own replicative enzyme which larger ones synthesize their own enzymes required for replication.
A few plasmids may integrate itself in a bacterial chromosome for replication. This integrated from is known as episome which may remain attached for numerous cell division but at some stage, it may exist in independent form.
1.3.    Plasmids classification :
1.3.1.    On the basis of the transfer of plasmid; it can be divided as
•    Conjugative plasmid: This plasmid is capable of transferring itself into bacteria by the process of conjugation. This function is specified by tra (transfer) and mob (mobilize) gene present on the plasmid. This promotes sexual conjugation between bacterial cells. Generally, this type of plasmids are of relatively higher molecular weight and are present as one to three copies per chromosome.

•    Non-conjugative plasmid: This type of plasmid cannot be transferred on their own but can be transferred only with the help of with conjugative plasmid. Both are present in the same cell and the mob region is functional. Most of the non-conjugative plasmids are of lower molecular weight and are present as multiple copies per chromosome.
1.3.2.    On the basis of the copy number
•    Relaxed plasmids: High copy number plasmids are known as relaxed plasmids and replication of such plasmids do not depend on replication of host chromosome.
•    Stringent plasmids: These of low copy number plasmids and replication is stringently controlled with replication of DNA.
There is an exception of R6K plasmid which is conjugative band and plasmid has a high molecular weight which belongs to the relaxed plasmid.
1.3.3.    On the basis of unimportant characteristic encoded by plasmid genes
•    Fertility or F plasmids: This plasmid has the ability to transfer i.e., the conjugate between bacterial cells. It carries only tra gene.
•    Resistance or R plasmid: It carries genes that confer resistance against one or more antibiotics like ampicillin, chloramphenicol etc.
Their spread through natural populations affects then treatment of bacterial infection and therefore it is important in clinical microbiology. For example RP4 in Pseudomonas
•    Col plasmids: This plasmid code for bacteriocins which is a type of protein that can kill bacteria. eg. ColEl of E. coli.
•    Degradative plasmids: This plasmid contains specific genes that allow the host bacterium to metabolize unusual molecules such as toluene, salicylic acid. eg. TOL of Pseudomonas putida.
•    Virulence plasmids: It provides pathogenicity to the host organism.
For example Ti plasmid of Agrobacterium tumefaciens which is responsible for crown gall disease in dicotyledonous plants. Plasmids are widespread in bacteria but are uncommon in another organism. The well-characterized eukaryotic plasmid is 2 mm circle which is found in streams of Saccharomyces cerevisiae. Thus cloning vector can be designed for yeast with the help of this plasmid.
1.3.4.    Size and copy number of plasmids :
These are important characteristics of plasmids as far as cloning is concerned. Plasmid should be smaller in size which can range from 1 kb for smallest to 250 kb for the largest. The larger ones are having low copy number and are stringent while smaller ones have multiple copies of 50 or more per cell and belong to relaxed plasmids.
1.3.5.    Partitioning and segregative sterility of plasmids
The low copy number plasmids are stable because of the presence of partitioning function. The par sequence which ensures stable maintenance of plasmid at each cell division. The loss of plasmid due to defective partitioning is known as segregative instability. High copy number plasmid can also have a par region like Col E1 but it is deleted in many Col E1 derived cloning vectors e.g., pBR322.

1.4.    Incompatibility of Plasmids
The inability of two plasmids to coexist in a single cell in absence of selection process is known as incompatibility of plasmids. Incompatibility can only be termed in the presence of the second plasmid. plasmids that are mutually incompatible belongs to the same incompatibility (including) group.
1.5.    Promoters and Directional Cloning :
A promoter is a nucleotide sequence that is present upstream of a gene and controls the expression of a gene. RNA polymerase binds specifically at this site and initiates transcription of a gene located downstream to the promoter. Therefore, for the formation of a product or expression of the gene, the foreign reign DNA should be inserted downstream of the promoter.
The foreign DNA can be digested with two different restriction enzymes which produce ends with different overhangs. Ligation of such molecule into plasmid vector can take place only in one orientation to give directional cloning. This directional cloning DNA molecule can be used for the construction of chimeric constructed in which foreign DNA with different overhangs can enter plasmid in only one direction.
Host range for plasmids :
Mostly the replication protein like DNA polymerases, DNA ligase, helicase and others are provided by the host cells, only a few proteins are synthesized by plasmids for their own replication. These replication proteins that are encoded by the plasmid are located near to ori. Therefore if another part of the plasmid is replaced by foreign DNA then also replication of plasmid will take place. Thus constructs from a plasmid can be created easily. For example the pUC family of the vector which has high copy number. PUC plasmid is consist of
•    Col E1 origin of replication
•    $\beta$-lactamase gene that confers resistance against ampicillin
•    inducible lac promoter (P lac) along with associated lac operon region
•    Lac I gene which codes for a lac repressor protein
•    Lac Z gene whose product is transcribed from lac promoter and produces an amino-terminal fragment of $\beta$ galactosidase protein.
•    Multiple cloning sites: it is a short stretch of DNA which contains a variety of restriction endonuclease. This is located just downstream from lac promoter and after few codons of lac Z gene.
Host range of plasmid depends on its ori region. In plasmid, Col E1 derived ori region containing plasmids have a restricted host range and can replicate only in entire bacteria E.coli, Salmonella etc. RP4 and RSF1010 have a broad host range. RP4 is a conjugative type of plasmid which can replicate in gram-negative bacteria and can be transmitted by conjugation. Therefore clone DNA molecules can be transferred into a wide range of genetic backgrounds.
On the other hand, RSF1010 are non-conjugative and can replicate in both gram-positive and gram-negative bacteria. Plasmids isolated from Staphylococcus aureus generally have a broad host range and can replicate in many gram-positive bacteria. These broad host range plasmids encode most of the proteins required for replication. Therefore it is required that their promoter and ribosome binding sites must be recognized in a diversity of bacterial families.

1.6.    Selection of Transformed cell:-
Strategy for selecting host cells that have been transformed with pBR322.
(1)     The transformation mixture, which contains three cell types, viz., Non transformed cells, cells with the intact original plasmid and cells with DNA cloned into the BamHI site of pBR322 is plated on complete medium with ampicillin.
(2)     The mixture is diluted beforehand to ensure separate colonies are formed on the agar. The nontransformed cells (Amps) are killed. The cells with the intact plasmid and cloned DNA-plasmid constructs are Ampr and therefore form colonies. Samples of the surviving colonies on the ampicillin plate are transferred to a plate with complete medium and tetracycline, keeping the same position of each colony on the second plate, i.e., Replica plating. Only cells with intact plasmids (Tetr) will form colonies in the presence of tetracycline.
(3)     The colonies that did not grow on the tetracycline plate (dashed circles) but grew on the ampicillin plate carry pBR322 with DNA that was cloned into the BamHI site. The colonies with cloned DNA inserts are picked from the original plate, pooled, and grown.

Biologically functional chimeric plasmids
Foreign DNA is inserted into plasmid DNA which forms hybrid DNA molecule known as a chimeric plasmid or recombinant plasmid. This insertion does not affect the property of the plasmid and hence the chimeric plasmid can propagate similarly on the original plasmid in the host organism. Several hundred copies of this plasmid can be found in bacteria. Hence any DNA sequence can be amplified or cloned via this method except inverted repeats, telomeres, centromere and origin of replication. For the amplification different promoters like inducible promoters, Plac, ptrP, T4,  SP6 can be used upstream to the gene.
The first biologically active recombinant or chimeric plasmid was synthesized in vitro by assembling different plasmids by Stanley Cohen, Herbert Boyer and Robert Helling. These were used to transform E. Coli. The Cells transformation is the process of uptake of foreign DNA from the environment by the recipient cell. Transformation is made possible by Ca+2 ion treatment and brief heat shock of 42°C which makes the recipient cell permeable but only 0.1% of Ca+2 treated bacteria became competent for transformation. These transformed bacteria can be selected by growing on antibiotics containing media. The chimeric plasmid must have been biologically functional in at least two aspects, one it should stably replicate within the host cell and secondly, it must confer resistance to antibiotics.
1.7.    Fusion Protein:-
Fusion proteins are proteins created through the joining of two or more genes that originally coded for separate protein. The translation of this fusion genes results in a single polypeptide with functional properties derived from each of the original protein.
A fusion protein cloning vector. The plasmid contains an ampicillin resistance (Ampr) gene as the selectable marker, a DNA sequence encoding the N-terminal segment of the E. Coli outer membrane protein (0mpF), a restriction endonuclease site (EcoRI) for cloning, and a truncated β-galactosidase gene (lacZ). The cloned gene is inserted into the EcoRI site. After transcription and translation, a hybrid protein is produced consisting of OmpF-target protein-LacZ.

1.8.    ARTIFICIAL PLASMID CLONING VECTORS :
1.8.1.    pBR322 :
p $\rightarrow$  Plasmid,  B $\rightarrow$  Bolivar,  R $\rightarrow$  Rodriguez, 322 $\rightarrow$  Plasmid number
1.    It was one of the first eukaryotic cloning vectors.
2.    It is 4361 bp length
3.    Bla gene encoded b lactamase which role play in ampicillin resistance.
4.    rep is an origin of replication from the pMB1 plasmid.
5.    rop gene encoding Rop protein associated with decrease copy no.

pUC :
p $\rightarrow$ Plasmid,  U $\rightarrow$ University, C $\rightarrow$ California
1.    Size is 2686 bp
2.    This vector is used in the broad spectrum because permits rapid visual detection of insert or no. Visual detection by vector contains a lacZ sequence and multiple cloning.
3.    pUC is advanced then pBR322 because of rep (ori) with a single mutation.
4.    BLa two point mutation
5.    Rop
The pBR322 plasmid is cleaved with restriction enzyme BamHI which is annealed or ligated by foreign DNA cleaved by BamHI restriction endonuclease, this creates a chimeric plasmid. These E. coli cells are treated with Ca+2 and 42° heat shock is given which makes the cell permeable for transformation. These cells are then plated on a Petri plate and incubated at 37°C overnight and colonies of AmpR bacteria is evident. TetR colonies can be determined by replica plating on tetracycline containing media. Only the colonies grown in both Petri plates possess plasmid with foreign DNA inserts. The proteins if any produced can be identified or quantified by affinity tag. The protein can be tagged with 6-histidine glutathione transferase (GST), mannose-binding protein (MBP), a Green fluorescent protein (GFP).
A map of pBR322 showing positions of the ampicillin resistance (amPR) and tetracycline resistance (tetR) genes, ori and some important restriction sites.