1. E.Coli AS
MODERN
VECTOR

2. E.Coli vectors:
Bacteria are the host of choice for DNA cloning. Among them E.coli occupies a prominent position as cloning and isolating DNA inserts for structural analysis is easiest in this host. The E.coli strain K12 is most commonly used. The initial cloning experiments are generally carried out in E.coli.

3. Properties of E.coli vector as a good host:
Easy to transform.
Support the application of recombinant DNA.
Free from elements that interfere in the recombination.
Lack active restriction enzyme.
Do not contain methylases that methylate the recombinant DNA and becomes resistant to some useful enzymes.

4. E.Coli supports several types of vectors, some natural and some constructed. And some of them are: Plasmids Bacteriophages Cosmids Phasmids Shuttle vectors Artificial chromosomes Phagemids

5. Plasmids:
A Plasmid is a DNA molecule, which is capable of independent replication and transmission.
Plasmids vary in size form about 1.0Kb to 250Kb.
An E.coli cell may contain up to 7 different kinds of plasmids.
Plasmids are circular and may exist independtly or become integrated into the bacterial chromosome.
There are three types of plasmids: F plasmids [responsible for conjugation], R plasmids [carry genes for resistance to antibiotics], Col plasmids [code for colicins, the proteins that kill sensitive E.coli cells ; they also carry genes]
Many plasmids are conjugative or transmissible as thay mediate DNA transfer through conjugation.

8. Bacteriophages These are bacteria's that attack viruses.
Several bacteriophages are used as cloning vectors, the most commonly used E.coli phages being lambda and M13 phages.
Phage vectors are tested on a appropriate bacterial lawn where each particle forms a plaque.

10. There are two advantage of phage vectors over Plasmid vectors:
They are more efficient than plasmids for cloning of large DNA fragments the la a largest cloned insert size in a lambda vector is just over 24Kb while that of plasmid vectors its less that 15Kb.
It is easier to screen a large number of phage plaques than bacterial colonies for the identification of recombinant vectors.

11. Cosmids
Cosmids are essentially plasmids that contain a minimum of 250bp of lambda DNA that includes the sequences from phage lambda genome – 1:the cor site [site having cohesive
ends] 2: sequences needed for
binding.
A cosmid has- 1: replication origin.
2: unique restriction site.
3: selectable markers from a plasmid.

13. Features of Cosmid vector :
They can be used to clone DNA inserts of up to 40kb.
They can be packed into a lambda particles, which infect host cell.
These vectors are amplified and maintained in the same manner as the contributing plasmid.

14. PHASMIDS:
Phasmids are lambda insertion vectors consisting of shortened linear lambda genomes containing DNA replication.
A phasmid vector contains several copies of the plasmid to make it longer than 38kb.
Phasmids are packed into lambda particles and used for infection of appropriate E.coli cells.

15. Shuttle vectors:
These vectors have been designed to replicate in cells of two different species, therefore they contain two regions of replication.
Created by recombinant techniques.
Some vectors are grown in prokaryotic species, usually E.coli.
A shuttle vector is designed to replicate in E.coli.

17. Shuttle vectors have been designed specifically to satisfy the initial cloning of DNA inserts in E.coli and subsequent functional tests in the species to which the DNA inserts belong.

18. Artificial chromosome
Artificial chromosome are circular or linear vectors that are stably maintained.
There are several type of vectors: bacterial artificial chromosome, yeast artificial chromosome [YAC], mammalian artificial chromosome [MAC], human artificial chromosome [HAC].
Of these vectors YAC is used for cloning in yeast, MAC and HAC are used in mammalian and human cells.

19. Phagemids
A plasmid vector that contains origin of replication from a phage, in addition to that of plasmid is known as phagemid.
The DNA insert can be integrated in a specific orientation by using two different restriction enzymes.
The DNA insert is then integrated in vitro in the double stranded vector.
The vector is then introduced into E.coli.
The recombinant DNA is propagated in E.coli as a plasmid using the origin of replication of a suitable vector.

20. Production of recombinant proteins in E.coli:
It can be done by two methods;
Transcriptional and Translational methods.
The site for the integration of the transgene is separated from the ribosome binding site by a sequence from the beginning of an E.coli gene known as transcriptional fusion.
The polypeptide obtained by the expression of the transgene is a hybrid and consist of short peptide encoded by the E.coli gene sequence followed by specified by the transgene, known as translational fusion.

21. Promoters for E.coli:
The promoter sequence has a great effect on the transcriptional rate. The promoters used in the modern era are:
The lac promoter: induced by IGPT, which provides a convenient control. This is the promoter of lac operon of E.coli.
The trp promoter: This promoter comes from E.coli trp operon.
The tac promoter: This promoter is a hybrid of lac and trp promoter.