Escherichia coli is a Gram-negative and rod-shaped bacteria that is mostly found in the lower intestine of humans and animals. It is a facultative anaerobe that means it can produce energy (ATP) in the presence of oxygen and switch to fermentation under anaerobic conditions.
Types of E. coli
There are two main types of E. coli, Commensal (non-pathogenic), and Pathogenic E. coli
Commensal (non-pathogenic) E. coli
Commensal strains of E. coli are a part of normal gut microbiota. They are useful in digestion and controlling the growth of other pathogenic bacteria in the gut. They also make vitamin B2 (riboflavin) and vitamin K2 (menaquinone). Some non-pathogenic strains of E. coli commonly used in the laboratory are E. coli K-12, E. coli BL-21, E. coli B.
Pathogenic E. coli
Pathogenic E. coli is further divided into two groups, intestinal pathogenic E. coli and extra-intestinal pathogenic E. coli.
Intestinal Pathogenic (enteric pathogens) E. coli
There are 7 types of intestinal pathogenic E. coli, and all of them cause diarrhoea like symptoms in humans. They can be transmitted through contaminated food and water.
Enteropathogenic E. coli (EPEC)
The presence of faecal matter in food or water can cause the spread of EPEC. It causes symptoms like watery diarrhoea with mucus, fever, and vomiting.
Enterohemorrhagic E. coli (EHEC)
This pathogenic E. coli can cause serious illnesses like haemolytic uremic syndrome (HUS) in humans. They produce a toxin called ‘shiga’ that damages the lining of the intestine and kidneys. Symptoms may include bloody diarrhoea, vomiting, and abdominal cramps.
Enteroinvasive E. coli (EIEC)
It invades the intestinal wall and causes symptoms like diarrhoea with a high fever. They are highly invasive and damage the intestinal wall, but they do not produce any toxins.
Enterotoxogenic E. coli (ETEC)
It produces a toxin that destroys the intestinal wall and is responsible for traveller’s diarrhoea. It produces two toxins, ST (heat-stable toxin) and LT (heat-labile toxin).
Enteroaggregative E. coli (EAEC)
This pathogenic E. coli can cause both acute and chronic diarrhoea in children of developing countries. They produce toxins that can destruct the intestinal wall. The 3 main toxins produced by EAEC are plasmid-encoded toxin (Pet), heat-stable toxin, and Shigella enterotoxin 1 (ShEt1).
Diffusely Adherent E. coli (DAEC)
It can cause acute diarrhoea in children. They are recognised by their diffused adherence patterns in which bacteria almost covers the entire epithelial cell.
Adherent Invasive E. coli (AIEC)
It is associated with Crohn’s disease. This pathogenic E. coli can adhere to the intestinal epithelial cells by Fim H and cell adhesion molecule 6.
Extraintestinal Pathogenic E. coli
They can cause serious infections like urinary tract infections, sepsis, pneumonia, and neonatal meningitis. They are of mainly four types.
Newborn meningitis E. coli (NMEC)
It causes meningitis in newborn infants. NMEC can evade the host defence mechanism to enter the blood-brain barrier.
Sepsis associated E. coli (SPEC)
This E. coli strain can cause serious infection in the blood (sepsis), and in serious cases, it can cause death. Many antibiotic-resistant strains of SPEC have emerged, especially multidrug-resistant strains.
Uropathogenic E. coli (UPEC)
This strain of E. coli is responsible for UTI (urinary tract infection) in humans. They have pili, fimbriae, and flagella on their surface, which help them to adhere to the epithelial cells.
Avian pathogenic E. coli (APEC)
This strain of E. coli can cause diseases like polyserositis, septicemia in chicken, and other avian species. Studies have shown that space between lungs and air sacs is an important site for APEC entry into the bloodstream, causing septicaemia. Factors responsible for the virulence of APEC are the presence of adhesions (pili, fimbriae) on the surface, resistance to bactericidal agents.
Features of E. coli that makes it a perfect model for molecular cloning and protein expression in the laboratory
- E. coli grows at a warm temperature of around 37.4C, which is easy to maintain in laboratories.
- It is a simple prokaryotic organism with well-understood genetics, which helps in easy genetic manipulation in E. coli.
- It has minimum and simple nutritional requirements. It needs a diet rich in carbon, nitrogen, and phosphorus for its growth.
- Being a facultative anaerobic bacteria, it can easily grow in the presence as well as in the absence of oxygen, thus it can be grown easily in culture flasks.
- The doubling time of E. coli is 20 minutes. It grows fast, which helps in the fast expression of the protein.
- The presence of plasmids in E. coli makes it an important tool in molecular cloning and protein expression. Plasmids are small double-stranded DNA molecules present in almost all bacterial cells (such as E. coli). The plasmid contains antibiotic-resistant genes, which can act as a selective marker in gene cloning. They are physically separated from bacterial DNA and can replicate independently of bacterial DNA. These plasmids can act as a vehicle (or vector) to introduce foreign DNA of our interest into the host bacterial cell.
Use of E. coli in Molecular Cloning
Cloning is a method in which multiple copies of a gene are produced by inserting the gene of interest into a suitable vector (mostly plasmid). The modified vector is then inserted into a competent host cell through a process called transformation. The most commonly used strains of E. coli for cloning are XL-1 blue and DH5α. As the bacterial host cell divides, the gene of interest also divides along with it, making multiple copies of the foreign inserted gene. Molecular cloning is useful in creating multiple copies of a gene for experiments like DNA sequencing, mutagenesis, protein expression, genotyping, etc.
What are Vectors?
A vector is a small piece of DNA molecule that can be maintained and easily replicated in a host organism (such as E. coli). Vectors are used to insert a foreign DNA (or gene) of interest with the help of endonucleases. A vector should have multiple cloning sites (MCS) that allow easy insertion of the foreign DNA fragment. MCS is a short DNA sequence with many restriction endonucleases sites. A few examples of cloning vectors are plasmids, cosmids, BAC (bacterial artificial chromosome), bacteriophages, etc.
What are Restriction Endonucleases?
These are enzymes that cleave the DNA into two fragments by breaking the phosphodiester bond within the polynucleotide chain. They cut the DNA at a specific nucleotide sequence (aka restriction sites). The action of these enzymes results in DNA fragments with either sticky or blunt ends. A few examples of restriction endonucleases are EcoRI, BamHI, and Hind III.
Use of E.coli in Recombinant Protein Production (RPP) by Pharmaceutical Companies
Recombinant protein is encoded by recombinant DNA, which is manipulated by genetic engineering to produce desired protein in large quantities. Recombinant proteins are used for the production of pharmaceutical products such as insulin, enzymes, recombinant hormones, thrombolytic drugs, etc. This process of making a recombinant protein is called recombinant DNA technology. E. coli has been commercially used as a factory to produce a recombinant protein of your choice. Due to less generation time and the ability to divide in large numbers, E. coli has been used as a suitable host for non-glycosylated protein production for therapeutic use. E. coli BL-21 and E. coli K 12 are two strains that have been commercially used for RPP (recombinant protein production); however, BL-21 is preferred for RPP as it lacks the enzyme proteases and produces less acetate than K-12, and thus it increases the biomass yield.
What is Recombinant DNA Technology?
As we know that proteins are required by our body for various functions, for example, the enzymes needed to digest food are protein in nature. We know that DNA is first transcribed into mRNA molecule which then translated into protein. In short, to make a recombinant protein first rDNA (recombinant DNA) has to be formed. Recombinant DNA technology is the method of bringing DNA from different species together, creating recombinant DNA, which would not otherwise be found normally in the genome.
Steps Involved in Recombinant Protein Production
- Introducing the gene of interest into an expression vector with help of restriction endonucleases. Expression vectors are usually plasmids or viruses designed especially for gene expression in host cells. An expression vector must-have features like an origin of replication, a promoter binding site, a ribosome binding site, a start codon, a termination codon, antibiotic-resistant gene (a selectable marker), and a multiple cloning site (MCS), where the gene of interest can be inserted.
- The expression vector containing the gene of interest is then transformed into a competent host cell (such as E. coli BL21). Transformation can be done by several methods like electroporation, heat shock treatment, etc.
- Once the expression vector has been transformed into the host cell, it will not start the translation of the desired protein until we induce it. Induction can be achieved by adding certain chemicals like IPTG (isopropyl B-d-1 thiogalactopyranoside). It triggers the transcription of recombinant DNA and hence the translation of desired recombinant protein.
- The recombinant protein thus formed can be isolated by the protein purification method. It is a process of isolating RPP from a complex mixture of cells, tissues, etc.
Difference Between Cloning Vector and Expression Vector
A cloning vector is used to carry a DNA fragment into a host cell and to make copies of it. Expression vectors are used for the expression of recombinant DNA into protein.
Role of E. coli in Biotechnology
If you are a biology student you must have heard of E. coli; it has been used in many biotechnology processes like molecular cloning, protein expression, DNA storage, biofuel production, etc. It has played a key role in many noble prize-winning findings such as DNA replication, operon system, genetic code, and genetically modified organism.
In Vaccine Production
We all are aware of the fact that vaccination is the most effective way of controlling the spread of infectious diseases. With the new technologies in science, we can protect ourselves from many infectious diseases through vaccination. Many vaccines have been generated using E. coli as a host such as a virus–like-particle (VLP) based vaccines. VLP vaccines are capable of inducing both innate and adaptive immune responses in humans. These vaccines contain viral structural protein but do not contain viral genetic material. E. coli has been used as a host for these viral-like particle-based vaccines. The first viral vaccine derived from E. coli was Hecolin, it is a recombinant VLP-based vaccine against the hepatitis E virus.
Bioremediation is a process of treating environmental pollutants by stimulating the growth of microorganisms that can break down the toxic pollutants and turn them into non-toxic compounds. Methomyl is a toxic chemical used in pesticides. It was observed in a research study that culturing E. coli in a medium containing methomyl can significantly lead to its degradation. It was later confirmed through many research studies that genes in plasmid and E. coli are responsible for the degradation of methomyl.
In Biofuel Production
The increasing cost of petroleum and depleting natural resources has made researchers explore new renewable sources of fuels. Biofuels like ethanol, biogas, and biodiesel have been produced by microbial activity. The use of microorganisms as a potential candidate for biofuel synthesis depends on their ability to produce biofuel at a faster rate with minimal cost. E. coli has been used as a potential generator of biofuel because of their well-studied gene regulation and growth metabolism. Under both aerobic and anaerobic conditions, E. coli can use carbon sources to produce biofuels. Genetic engineering and Synthetic biology have made it possible to generate new biosynthetic pathways and incorporate these in E. coli for optimal production of biofuels.
Genetic Engineering of E. coli for Better Production of Ethanol
Bioethanol is one of the most commercially used biofuels and is majorly produced from olignocellulose (lignin, hemicelluloses, and cellulose). Hexose sugars, released after hydrolysis of hemicelluloses, are converted into ethanol by certain yeast and bacteria through fermentation. S. cerevisiae is, however, not capable of fermenting pentose sugar into ethanol. In this case, E. coli can be used as a biofuel producer as it can ferment both pentose and hexose sugar under anaerobic conditions. E. coli can produce ethanol by a pathway in which under anaerobic conditions, it metabolizes 1 glucose molecule into 2 molecules of formate, 2 molecules of acetate, and 1 molecule of ethanol. Ingram et al have made some changes in this endogenous pathway of E. coli to produce ethanol in high quantity by inserting 2 genes, pdc, and adhB from Z.mobilis. The introduction of these 2 gens into E. coli has improved ethanol production by 95%.
For Storage of DNA Sequences
E. coli can be used for the storage of DNA sequences from other organisms like humans. E. coli containing the DNA sequence of other organisms can be stored in deep freezers for a very long time. The researcher can retrieve these DNA sequences by thawing the E. coli cells at 37°C (gut temperature) and then treating them with endonucleases. E. coli can also produce multiple copies of the inserted DNA fragment by culturing the cells in liquid media and keeping them in a shaker incubator. This causes E. coli cells to divide rapidly and thus producing multiple copies of the desired inserted DNA fragment.
In E. coli Based Biosensors
Pollution has now become a major global concern. We need new devices like biosensors, which can easily detect the presence of hazardous pollutants in the environment. Biosensors are devices that use biological components to detect any physiochemical changes in the surrounding. These devices are consist of two components, biological, and transducer or detector. The biological component can be any biological elements like cells, organisms, tissues, etc. A transducer converts signals, resulting from the interaction of the analyte with biological components, into physiochemical signals (electrochemical, piezoelectric signals). Researchers have made E. coli biosensors to detect the presence of 3-phenoxybenzoic acid (3-PBA). 3-PBA is a primary metabolite of the pyrethroids class of insecticides. Pyrethroids are neurotoxins and can cause serious illnesses in humans like skin irritation, nausea, suppressed immune system, and prolonged exposure to pyrethroids may lead to cancer. The presence of 3-PBA in urine and plasma can be easily detected using a whole-cell biosensor. It is based on competitive ELISA and is consists of whole E. coli cells displaying anti -3-PBA antibodies on their surface. When these engineered E. coli cells are mixed with 3-PBA protein conjugate, cross-linking occurs, which can be easily detected visually. When a sample containing free 3-PBA is added to this mixture, it competes with the crosslinking, which results in a change in output. Thus the presence of 3-PBA in the urine or plasma samples of humans can be easily detected.