What are the processes of gene transfer between bacterial cells?

Bacteria do not have an obligate sexual reproductive stage in their life cycle, but they can be very active in the exchange of genetic information. The genetic information carried in the DNA can be transferred from one cell to another; however, this is not a true exchange, because only one partner receives the new information. In addition, the amount of DNA that is transferred is usually only a small piece of the chromosome. There are several mechanisms by which this takes place. In transformation, bacteria take up free fragments of DNA that are floating in the medium. To take up the DNA efficiently, bacterial cells must be in a competent state, which is defined by the capability of bacteria to bind free fragments of DNA and is formed naturally only in a limited number of bacteria, such as Haemophilus, Neisseria, Streptococcus, and Bacillus. Many other bacteria, including E. coli, can be rendered competent artificially under laboratory conditions, such as by exposure to solutions of calcium chloride (CaCl2). Transformation is a major tool in recombinant DNA technology, because fragments of DNA from one organism can be taken up by a second organism, thus allowing the second organism to acquire new characteristics.

Transduction is the transfer of DNA from one bacterium to another by means of a bacteria-infecting virus called a bacteriophage. Transduction is an efficient means of transferring DNA between bacteria because DNA enclosed in the bacteriophage is protected from physical decay and from attack by enzymes in the environment and is injected directly into cells by the bacteriophage. However, widespread gene transfer by means of transduction is of limited significance because the packaging of bacterial DNA into a virus is inefficient and the bacteriophages are usually highly restricted in the range of bacterial species that they can infect. Thus, interspecies transfer of DNA by transduction is rare.

Conjugation is the transfer of DNA by direct cell-to-cell contact that is mediated by plasmids (nonchromosomal DNA molecules). Conjugative plasmids encode an extremely efficient mechanism that mediates their own transfer from a donor cell to a recipient cell. The process takes place in one direction since only the donor cells contain the conjugative plasmid. In gram-negative bacteria, donor cells produce a specific plasmid-coded pilus, called the sex pilus, which attaches the donor cell to the recipient cell. Once connected, the two cells are brought into direct contact, and a conjugal bridge forms through which the DNA is transferred from the donor to the recipient. Many conjugative plasmids can be transferred between, and reproduce in, a large number of different gram-negative bacterial species. Plasmids vary in size, from a few thousand to more than 100,000 base pairs; the latter are sometimes called megaplasmids.

The bacterial chromosome can also be transferred during conjugation, although this happens less frequently than plasmid transfer. Conjugation allows the inheritance of large portions of genes and may be responsible for the existence of bacteria with traits of several different species. Conjugation also has been observed in the gram-positive genus Enterococcus, but the mechanism of cell recognition and DNA transfer is different from that which occurs in gram-negative bacteria.

The transfer of genes between species is called gene modification, and the new organism created is called a transgenic

Genetic Modification: Bacteria Producing Human Insulin

Application:

•  Gene transfer to bacteria using plasmids makes use of restriction endonucleases and DNA ligase

    
The process of gene transfer can be summarised in four key steps:

1. Isolation of gene and vector (by PCR)
2. Digestion of gene and vector (by restriction endonuclease)
3. Ligation of gene and vector (by DNA ligase)
4. Selection and expression of transgenic construct

Step 1:  Isolating gene and vector

• DNA can be isolated from cells by centrifugation – whereby heavier components such as nuclei are separated
• The gene of interest can then be specifically amplified via the polymerase chain reaction (PCR)
• Gene sequences can also be generated from mRNA using reverse transcriptase – these DNA sequences (cDNA) lack introns
  
  
• A vector is a DNA molecule that is used as a vehicle to carry the gene of interest into a foreign cell
• Bacterial plasmids are commonly used as vectors because they are capable of autonomous self-replication and expression
• These plasmids may be modified for further functionality (e.g. selection markers, reporter genes, inducible expression promoters) 
• Other types of vectors include modified viruses and artificial chromosomes

Common Features of a Typical Plasmid Vector

Step 2:  Digestion with Restriction Enzymes

• In order to incorporate a gene of interest into a vector, both must be cut with restriction enzymes at specific recognition sites
• Restriction enzymes cleave the sugar-phosphate backbone to generate blunt ends or sticky ends (complementary overhangs)
• Scientists will often cleave the vector and gene with two different ‘sticky end’ restriction endonucleases (double digestion) to ensure the gene is inserted in the correct orientation and to prevent the vector from re-annealing without the desired insert

‘Sticky End’ vs ‘Blunt End’ Restriction Enzymes

Step 3:  Ligation of Vector and Insert

• The gene of interest is inserted into a plasmid vector that has been cut with the same restriction endonucleases
• This occurs because the sticky ends of the gene and vector overlap via complementary base pairing
• The gene and vector are then spliced together by the enzyme DNA ligase to form a recombinant construct
• DNA ligase joins the vector and gene by fusing their sugar-phosphate backbones together with a covalent phosphodiester bond

Formation of a Recombinant Construct

Step 4:  Selection and Expression

• The recombinant construct (including the gene of interest) is finally introduced into an appropriate host cell or organism
• This process can be achieved in a variety of ways and is called transfection (for eukaryotes) or transformation (for prokaryotes)
• Antibiotic selection is commonly used in order to identify which cells have successfully incorporated the recombinant construct
• The plasmid vector contains an antibiotic resistance gene, so only transgenic cells will grow in the presence of antibiotic
• Transgenic cells, once isolated and purified, will hopefully begin expressing the desired trait encoded by the gene of interest

  What are the methods of gene transfer between bacteria?

  There are three “classical" methods of DNA transfer in nature: bacterial conjugation, natural transformation, and transduction (von Wintersdorff et al., 2016). Via HGT, exogenous DNA can be transferred from one bacterium to another even if they are only distantly related (Chen et al., 2005; Burton and Dubnau, 2010).

  What are 3 methods of transformation of bacteria?

  Key steps in the process of bacterial transformation: (1) competent cell preparation, (2) transformation of cells, (3) cell recovery, and (4) cell plating.

  What are the 5 steps of bacterial transformation?

  Step [1] Remove Plasmid from bacteria cell..

  Step [2] Isolate the gene of interest..

  Step [3] cut open plasmid with restriction enzymes, leaves "Sticky ends"..

  Step [4] insert gene of interest..

  Step [5] Insert the Plasmid with Recombinant DNA into a new bacterium..

  Step [6].

  What is the process of transformation in bacteria?

  Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. It was first reported in Streptococcus pneumoniae by Griffith in 1928. DNA as the transforming principle was demonstrated by Avery et al in 1944.