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  Section: General Biotechnology / Genes & Genetic Engineering
 
 
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Techniques of Genetic Engineering

 
     
 

Expression of Cloned DNA
Factors taken into account for expression are :

(i)

Supply of prokaryotic promoter for expression of eukaryotic genes,

(ii)

Supply of ribosonal binding sites for the cloning vector,

(iii)

Removal of introns from mRNA obtained from eukaryotic genes (see sections cDNA Clone bank and Gene bank (Genomic Library))

(iv)

Inhibition of gene responsible for degradation of foreign protein within the bacterium.


The genes of eukaryotes differ in different ways from those of E.coli. Therefore, the eukaryotic genes must be modified in such a way that they could resemble the prokaryotic, genes and express in E.coli. To begin with transcription in prokaryotes the binding of bacterial RNA polymerase to a promotor region of the DNA immediately before (or upstream of) the gene is essential.

Shine-Dalgarno sequence
Prokaryotic mRNA, have a sequence upstream from the initiation codon that play a role in attachment of the 30S ribsomal subunit to the mRNA. This sequence (about 3-9 bases long) is about 3-12 bases upstream from the initiation codon which is also complementary to a sequence near the 3' terminus of 16 S rRNA (Shine and Dalgarno, 1975). The name of this sequence was coined as the 'Shine-Dalgarno sequence' after the name of the discoverer. For the expression of eukaryotic gene within E. coli, presence of a Shine-Dalgarno sequence in bacteria is necessary.

Expression vectors
In cDNA, translation is correctly started at the universal initiation codon, AUG. To overcome the problem of time consumption in adding a Shine-Dalgarno promoter sequence and initiation codon to every foreign gene, the expression vector has been built up. It contains a strong promoter, the activities of which are controlled by temperature, concentration of specific inducer or repressor and a Shine-Dalgarno sequence. In addition to promoter, the expression vectors also have restriction sites, origin of replication and marker genes (for resistance against the specific antibiotics).

Therefore, it is necessary to replace the eukaryotic promoter with  a prokaryotic promoter in order to carry out transcription of an eukaryotic gene. Hence, for the construction of expression vectors a number of bacterial promoters have been used, for example, promoter or lac or trp operon from E. coli and b-lactamase promoter from pBR322 plasmid.

The lac promoter is a hybrid promoter constructed between elements of the trp and lac promoters. These promoters are regulated by their corresponding repressors. The lac operon is negatively regulated by the lac repressor and can be depressed by addition of an inducer i.e. isopropyl b-D-thiogalactosidase (IPTG). It is positively regulated by the catabolic activator protein (CAP) complexed with cyclic AMP pathway. Similarly, the trp operon encodes 5 enzymes for the biosynthesis of tryptophan. The complex formed between the trp repressor and tryptophan negatively control the trp operon. When the amino acid is in abundant supply the genes responsible for its biosynthesis are repressed.

 

Content

Gene cloning in prokaryotes

 

Isolation of DNA to be cloned

 

Insertion of DNA fragment into vector 

 

 

Use of restriction Linkers

 

 

Use of homopolymer tails

 

Transfer of recombinant DNA into bacterial cells

 

Selection of clones

 

 

Colony hybridization techniques

 

 

In vitro translation technique

 

 

Immunological tests

 

 

Blotting Techniques

 

Recovery of cells

 

Expression of cloned DNA

 

 

Shine-Dalgano sequence

 

 

Expression vectors

Gene cloning in eukaryotes

 

Plant cells

 

 

Yeasts

 

 

Filamentous fungi

 

 

Agrobacterium plasmids

 

 

Plant cell transformation

 

 

Plant cell transformation by ultrasonication

 

 

Liposome mediated gene transfer

 

Animal cell  

 

 

Animal viruses

 

 

Electroporation

 

 

Particle bombardment

 

 

Microinjection

 

 

Direct transformation

Site directed mutagenesis

 

Methods of mutagenesis


However, the eukaryotic proteins are not always stable in prokaryotic cells; it is disintegrated by the bacterium. Protection of protein is necessary. When a foreign gene is inserted in a prokaryotic gene, a hybrid polypeptide is produced. It contains a part of the prokaryotic product attached to N-terminus of the foreign polypeptide. The extra sequence of N-terminus may stabilize the polypeptide and may also interfere in the right functioning of the foreign protein. Therefore, it is necessary to separate the hybrid protein by chemical cleavage at methionine.
 
     
 
 
     



     
 
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