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What chemicals will kill P1 Phage but not E. coli?

What chemicals will kill P1 Phage but not E. coli?


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I'm working with E. coli and P1 Phage. I'm wondering if there is some chemical agent that will kill or disable P1 but leave E. coli untouched? It is not enough to just prevent infection. It must destroy the Phage.


New material kills E. coli bacteria in 30 seconds

Researchers in Singapore have developed a new material that can kill the E. coli bacteria within 30 seconds.

Every day, we are exposed to millions of harmful bacteria that can cause infectious diseases, such as the E. coli bacteria. Now, researchers at the Institute of Bioengineering and Nanotechnology (IBN) of Agency for Science, Technology and Research (A*STAR), Singapore, have developed a new material that can kill the E. coli bacteria within 30 seconds. This finding has been published in the peer-reviewed journal, Small.

"The global threat of drug-resistant bacteria has given rise to the urgent need for new materials that can kill and prevent the growth of harmful bacteria. Our new antimicrobial material could be used in consumer and personal care products to support good personal hygiene practices and prevent the spread of infectious diseases," said IBN Executive Director, Professor Jackie Y. Ying.

Triclosan, a common ingredient found in many products such as toothpastes, soaps and detergents to reduce or prevent bacterial infections, has been linked to making bacteria resistant to antibiotics and adverse health effects. The European Union has restricted the use of triclosan in cosmetics[1], and the U.S. Food and Drug Administration is conducting an on-going review of this ingredient [2].

Driven by the need to find a more suitable alternative, IBN Group Leader Dr Yugen Zhang and his team synthesized a chemical compound made up of molecules linked together in a chain. Called imidazolium oligomers, this material can kill 99.7% of the E. coli bacteria within 30 seconds aided by its chain-like structure, which helps to penetrate the cell membrane and destroy the bacteria. In contrast, antibiotics only kill the bacteria without destroying the cell membrane. Leaving the cell structure intact allows new antibiotic-resistant bacteria to grow.

"Our unique material can kill bacteria rapidly and inhibit the development of antibiotic-resistant bacteria. Computational chemistry studies supported our experimental findings that the chain-like compound works by attacking the cell membrane. This material is also safe for use because it carries a positive charge that targets the more negatively charged bacteria, without destroying red blood cells," said Dr Zhang.

The imidazolium oligomers come in the form of a white powder that is soluble in water. The researchers also found that once this was dissolved in alcohol, it formed gels spontaneously. This material could be incorporated in alcoholic sprays that are used for sterilization in hospitals or homes.

E. coli is a type of bacteria found in the intestines of humans and animals, and some strains can cause severe diarrhea, abdominal pain and fever. Such infection is contagious and can spread through contaminated food or water, or from contact with people or animals. Good hygiene practices and proper food handling can prevent


Bacteriophage Vectors for E.Coli | Genetics

The cloning of single genes is usually best carried out using plasmids, since the insert will rarely be larger than about 2 kb. However, for cloning of larger pieces of DNA (e.g., during gene library construction) these plasmids are not suitable as larger inserts increase the plasmid size, making the transformation inefficient. Large DNA molecules can be injected in host bacterial cell by viral particles (bacteriophages). Commonly used bacteriophages are M13, f1, fd and lambda (λ) phage.

(i) Phage Lambda (λ) as a Vector:

A commonly used vector is that of the lambda (λ) phage. Bacteriophage λ, which infects E. coli cells, can be used as cloning vector. DNA of λ phage is 48.5 kb in length. At its ends are the cos (cohesive) sites, which consist of 12 bp cohesive ends. The cos ends allow the DNA to be circularized in the host cell.

For the cloning of large DNA fragments, up to about 20 kb, much of the nonessential lambda DNA is removed and replaced by the insert (desired or ta. The recombinant DNA is then packaged within viral particles in vitro, and these are allowed to infect bacterial cells (E. coli) which have been plated out on agar.

Once inside the bacterial cells, the recombinant viral DNA is replicated. All the genes needed for normal lytic growth are still present in the DNA and so multiplication of the virus takes place by cycles of cell lysis and infection of surrounding cells. It gives rise to plaques of lysed bacterial cells on a background, or lawn, of bacterial cells. Cloned DNA can be recovered from the viruses in these plaques.

There are following two types of lambda vectors phages which differ in size of DNA they accept:

(a) Lambda replacement vectors and

(b) Lambda insertion vectors.

(a) Lambda Replacement Vectors:

Lambda replacement vectors contain a restriction site for phage propagation in suitable bacterial host. Remaining part of the lambda genome is removed and is replaced by foreign DNA. Ligation is performed at a ratio of arms to target DNA that favours the formation of very long concatemers. The concatemers are multiple length copies with multiple replication complexes and forks. In each of concatemers, vector and target molecules are interspersed.

A number of so-called replacement vectors have been developed from phage λ, examples include EMBL4, EMBL3, λ DASH, etc. In most replacement vectors, the internal region that is replaced by the target contains a gene that renders the phage inviable (dead) in an appropriate E. coli host Vector molecule containing target DNA can be selected by infecting host. Recombinant phage in which the internal region is replaced by target DNA is viable and are mostly used for cloning eukaryotic DNA fragments.

(b) Lambda Insertion Vectors:

When cloning into an insertion vector, the phage DNA is cleaved with a restriction enzyme that cuts it only once, and the target is inserted into this site No phage DNA is removed, therefore, much smaller sized target DNA can be inserted.

In commonly used vector phage λ gt 10, the Eco II cloning site is in a gene which is deleterious to phage replication in certain host strains. This allows selection against non-recombinant phage.

(ii) Filamentous Phages as Cloning Vector:

It includes Ff class of filamentous phages, including strains f1, fd and M13, which infect E. coli cells. These Ff virions are long and thin and contain a closed loop of single- stranded DNA. Because the phages readily accept inserts of foreign DNA and they supply one strand of that DNA in an easily isolated form, vectors based on Ff phages have become standard choice of biotechnology.

a. M13 Bacteriophage:

The M13 is a filamentous bacteriophage of E. coli. It is 870 nm long and 6 nm wide. It has a protein coat (the capsid) which is made up of three kinds of capsomeres. This filamentous single stranded DNA phage infects the bacterial cell by adsorbing to and entering through a pilius. M13 phages only infect F + or Hfr cells, not F – cells.

The M 13 phage particles contain a 6.7 kb circular single stranded DNA. After infection of a sensitive E. coli host, the complementary strand is synthesized, and the DNA replicated as a double standard circle, the replicative form (RF), with about 100 copies per cell.

Further M13 phages do not lyse the host cells (to release progeny phage and bacterial DNA from the ruptured bacterial host cells) like lambda phage during the lytic cycle. Instead of this, the progeny M13 viruses/phages are extruded through the layers of the plasma membrane and cell wall without major interference with cell growth.

Infected bacterial cells will continue to grow and extrude thousands of progeny virus particles (i.e., up to 1000 per cell generation), each containing a single- stranded genome to the medium. Since the virus particles are very small in comparison of the host bacteria, the host cells can be removed by low-speed centrifugation.

The virus particles can then be collected from the supernatant suspension by high-speed centrifugation and their single-strand DNA molecules can be isolated by single phenol-chloroform extractions. The DNA strand of the virus is always packaged it is called the “+” or plus strand (its complement is the “-” or minus strand).

The packaged “+” strand of the phase has the same “sense” as the mRNA, i.e., nucleotide triplets of + DNA strand of M13 phage correspond to the mRNA codons, but with T in place of U.

Packaging of single strands of phage DNA in progeny phage provides a neat biological purification of single-strand DNA. Importantly, this statement holds true for a foreign gene cloned in the viral chromosome just as for the phage genes themselves. This property of M13 phase has been exploited for its use as a vector.

Lastly, phage M13 is not used as a primary vector to clone new DNA targets, but fragments are normally sub-cloned into M13RF using standard plasmid methods when the single-stranded form of a fragment is required.

1. Genetic organisation of wild type M13 bacteriophage:

The DNA molecule of M13 phage is single-stranded and circular. It is 6407 bases long having 10 closely packed genes. All these genes are essential for the replication of the phages. There is a segment of 507 base-long intergenic sequence (IS) which contains origin of replication (OR).

The IS containing region of viral genome is manipulated for cloning without disrupting the origin of replication. Hence, the wild M13 phage has limited use in gene cloning experiments. The size of the phage particle is decided by the size of the phage DNA. Upto six times the normal length of M13 phage DNA can be packaged.

2. Construction of M13 based vectors:

In M13 phage DNA, the intergenic sequence is the only region which can be manipulated for gene cloning. As this region has only two restriction sites (Asa I and Ava II), wild type phage is not an efficient vector. However, the intergenic sequence can be modified to introduce additional restriction sites.

A few such vectors are discussed below:

M13mp1 and M13mp2:

The gene lac Z is introduced into the wild type intergenic sequence to get the M13 mpl phages. This step produces blue plaques on X-gal agar plates. The lac Z gene does not have any restriction site. However, it has a hexanucleotide sequence, GGATTC near the start. If the second G residue is substituted by an A residue this sequence becomes an Eco R1 site, i.e., GAATTC. This type of conversion is done by in vitro mutagenesis.

Now this phage is called M13 mp2. The lac Z’ gene now has a slightly altered base sequence. Due to this change, the β-galactosidase enzyme which is encoded by lacZ gene has amino acid asparagine instead of aspartic acid (amino acid) in the fifth position. Such a change (due to mutation) does not affect the activity of the β-galactosidase enzyme. This can be tested by X-gal agar where the plaque gives a blue colour.

Gene lac Z of E.coli encodes the N-terminal a-peptide of the enzyme (3-galactosidase which is responsible for hydrolysis of lactose into galactose and glucose.

The M13 mp2 is the simplest cloning vector derived from M13 phage. In the unique Eco RI site of M13 mp2, any foreign DNA with Eco RI sticky ends can be inserted. This insertion inactivates lac Z gene and is called insertional inactivation. Such recombinant phages fail to produce blue plaques on X-gal agar, instead, they produce clear plaques.

The M13 mp7 is a derivative of MB mp2. When a polylinker is inserted into the Eco RI site of lac Z’ gene, the M13 mp7 becomes M13 mp7. The polylinker is designed in such a way that it does not inactivate the lac Z’ gene.

When the vector phage M13 is cut with Eco RI, Bam HI, Sal I or Pst I, the entire polylinker or a part of it is excised and form sticky ends. Foreign DNA with the corresponding sticky ends is inserted to produce recombinant M13 mp7. Thus, M13 mp7 is a more complex vector having four possible insertional sites.

Insertion of foreign DNA tends to inactivate the lac Z gene and the production of galactosidase enzyme is prevented. This is shown by the formation of clear plaques on X-gal agar by the recombinant phage DNA.

3. M13-Plasmid Hybrid Vectors:

The hybrid vectors contain components from both plasmids and phage chromosomes. These vectors replicate in E.coli as normal double-stranded plasmids until a helper phage is provided. After the addition of the “helper” phage, they switch to the phage mode of replication (i.e., rolling circle replication) like that observed for phage ф×l- and package single strands of DNA in phage particles.

The helper phage is a mutant that replicates its own DNA inefficiently, but provides viral replication enzymes and structural proteins for the production of plasmid DNA molecules that are packaged in phage coats.

(b) pUC 118 and pUC 119 Cloning Vectors:

The phage-plasmid “hybrid” vectors pUC118 and pUC119 are a pair of vectors that are essentially identical except that the regions into which foreign DNAs are inserted are present in opposite orientation (i.e., turned end-to-end relative to the rest of the genes of the vector.

Thus, if a foreign DNA is inserted into a specific restriction site in both vectors, one vector will package the complementary strand of the gene. Therefore, both strands of the gene can be isolated, sequenced, subjected to site-specific mutagenesis, and so on.

The vectors were designated pUC for plasmid, University of California (where the first pUC vectors in the series were constructed by J. Messing and J. Vieira 1987) and 118, 119 to distinguish from earlier members (i.e., lower numbers) of the series.

Vectors pUC 118 and pUC 119 differ from earlier vectors in the pUC series by the addition of are origin of replication from phage M13. This permits pUC118 and pUC119 to replicate either (1) as a double standard plasmid in the absence of “helper” phage or (2) as a single-stranded DNA that is packaged in M13 phage coats and extruded from the cell in the presence of “helper” phage (most commonly an M13 derivative called K07).

In the absence of “helper” phage, replication is controlled by the plasmid origin of replication. The pUC vectors were derived from an early plasmid cloning vector called pBR322 by a series of direct modifications.

The pUC vector such as PBR322 contain an origin of replication initially present in plasmid Col E1. In the presence of helper” phage replication of pUC118 and pUC119 is directed by the M13 phage origin of replication that has been added to the plasmids.

Certain important features of the pUC11S and pUC119 cloning vectros are the following:

1. They are present as small, supercoiled, covalently closed circular DNA. Due to this feature they are easily isolated and manipulated in ‘ vitro.

2. They carry the amp” gene as a selectable marker. Thus, only bacteria harbouring a plasmid will grow on medium containing the antibiotic amplicillin.

3. They carry high copy number, up to 5000 copies per bacterium. Thus, there would be large yields of DNA from small cell cultures.

4. They carry a polycloning region which have a variety of restriction enzyme cleavage sites. Thus many different types of restriction fragments can be inserted without modification.

5. The polycloning region interrupts the coding region of the 5′ end of E. coli lac Z gene. Thus, colonies harbouring plasmids with foreign DNA inserts can be distinguished from those carrying plasmids with no insert by a simple colour test.

6. The lac Z gene is under the control of the lac promoter. Thus, genes inserted in frame (codons in proper reading frame) can be expressed to produce β-galactosidase- foreign protein fusion products.

7. They carry a plasmid origin of replication. Thus, replication of the vector DNA produces large numbers of double-stranded plasmid DNAs in the absence of “helper” phage.

8. They carry a phage M13 origin of replication. Thus production of single-stranded DNA and packaging of this DNA in phage coats take place in the presence of “helper” phage.

9. The polycloning regions are present in pUC118 and pUC119 in opposite orientations. Thus, if pUC118 packages one strand of a cloned gene, pUC119 will package the complement strand.

Different members of the pUC vectors series contain different, but related, sets of restriction enzyme cleavage sites. The polycloning regions of pUC118 and pUC119 contain 10 clustered restriction enzyme cleavage sites. Some of these sites are substrates for two or more different restriction enzymes.

The utility of pUC is greatly increased by a simple colour test that allows one of distinguish cells harbouring plasmids with foreign DNA inserts from those harbouring plasmid with no insert. The basis of this colour indicator test is the functional inactivation of the 5’ segment of the lac Z gene present in the vector by the insertion of foreign DNA into the polycloning region.

The pEMBL8 Cloning Vehicle:

The pEMBL8 cloning vehicle is constructed by transforming a 1300 bp fragment of the genome of M13 phage into the pUC8 plasmid. This piece of M13 genome has the signal sequence recognised by the enzymes that convert the dsM13 DNA into a ssDNA molecule. So the pEMBL 8 molecules are also converted into single-stranded DNA molecules and these will be released as infective phage particles.

When pEMBL 8 vector is used, the E. coli cells should also be infected with an intact M13 particle. This will act as a helper phage by providing necessary enzymes and phage coat proteins. Since pEMBL 8 is derived from pUC8 it has the polylinker at the necessary lac Z’ gene.

Hence this plasmid vector has the following advantages of pUC8 plasmids and that of the M13 phages:

1. Foreign DNA fragments with two different sticky ends can be inserted at the multiple cloning site on the lac Z’ gene.

2. Recombinant pEMBL 8 genome can be packaged into the capsid of M13 phage.

3. The phage particles containing the recombinant pEMBL 8 genome are as efficient as the wild type M13 phages in their ability to infect E. coli cells and then in expression of the foreign gene.

4. Recombinant pEMBL 8 genome can be easily screened using the medium containing X-gal.


Swan song for antibiotics? Can phage therapy and gene editing fill the gap?

It’s time to face the music: the golden age of antibiotics is over.

Humans have been in an endless war against bacteria — indeed it can kill us — and for decades antibiotics have been our only real weapon against them. They revolutionized medicine in the 20th century, and have together with vaccination led to the near eradication of many diseases in the developed world.

But despite their amazing success, and they’ve had a miracle like run, drug resistant superbugs like MRSA, CRE and VRE are slowly winning. Every year more and more people die from drug resistant infections as we inch closer back to the pre-penicillin days. Their effectiveness and easy access led to overuse, especially in live-stock raising, prompting bacteria to develop resistance. This led the World Health Organization last year to classify antimicrobial resistance as a “serious threat [that] is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country”

There is a lot of blame to be shared for this reversal: doctors prescribing antimicrobials for the common cold, patients demanding the drugs for the flu, governments de-incentivizing the development of new antibiotics and pharmaceutical companies deeming unprofitable.

Regardless of who might be blamed, this current situation was bound to happen our haphazard behavior simply hastened the arrival of this day. Whenever the goal is to kill a living organism resistance is inevitable. It’s how nature works: natural selection. In this case an antibiotic kills off all susceptible bacteria, but a hardy few resistant to the drug because of random mutations survive, and eventually thrive. On the verge of wiping out a problem, it roars back even worse than how it started.

This is how all life responds to stressors. We adapt. It shows up in the plant world as well. Look at the firestorm debate over herbicide resistant weeds in agriculture. Whether a farmer uses organic approved herbicides, conventionally bred ones (such as conventionally bred sunflower crops) or crops genetically engineered to resist glyphosate, mutant weeds survive and adapt and supersedes develop. It’s the law of nature. Yes, we can change up herbicides or create new ones, but the process then begins again. That’s true about antibiotics as well. So even if we create a new class of antibiotics, resistance will eventually follow, no matter how responsible we are with using it.

In this light, it’s clear that terms like “superbugs” and “superweeds” are misnomers because these organisms aren’t special in any way, they are simply doing exactly what nature intends them to do.

What compounds the challenge for antibiotics is that bacteria appear to be able to evolve resistance faster than any other type of organism. Conjugation (bacterial sex) allows bacteria to share resistance genes with not only members of their species, but across species and even across bacterial type (i.e. gram positive to gram negative). This is called horizontal gene transfer and allows advantageous traits to be spread among members of the current generation, instead of in future generations. Furthermore, some evidence suggests that bacteria can also sense when their back is against the wall and mutate their genome at faster rates in a last ditch effort for one of their descendants to mutate enough to develop resistance to the antibiotic.

But Darwin aside, it’s in our best interest to move beyond the era of antibiotics. Using antibiotics to treat an infection is like using a sledge hammer to pound in a nail. Sure the nail will get pounded in but there’s also going to be a large hole in your wall. Even the most narrow spectrum of antibiotics drastically affect our normal flora in ways that can have far reaching consequences on our physiology.

Phage therapy

What we need in our war against microbes are biocidal agents that are effective but also have more species specificity. One strategy in the works involves turning to the natural enemy of the bacteria, the virus for help. In other words the enemy of my enemy is my friend.

Viruses don’t just infect humans, in fact every species on the planet has numerous species of virus that can infect them. When a virus infects a bacteria, scientists have a special name for them: bacteriophage, or just phage. In bacteria, as they do in humans, viruses work by gaining access to the interior of a cell and manipulating the host’s cell machinery (i.e. enzymes, ribosomes etc) into making more copies of the virus. At a certain point, the number of copies of a virus induces the cell to burst releasing sometimes thousands of new copies of the virus, which can then go on to infect another cell and the process repeats.

Corrupting this process to fit human needs in the fight against antibiotics has actually been going on since the early days of the cold war. While the U.S. was investing in antibiotics, the Soviets were developing what is now called phage therapy. In fact in places like the country of Georgia, Russia and Poland phage therapy is still widely used today to treat infections.

One of the major benefits of phage therapy over traditional antibiotics is the specificity a phage has for its host. For the most part, a phage that infects an E. coli will not be able to infect another bacteria type, like a commensal Staphylococcus. There is even some evidence that phages are strain specific meaning we can develop phages that can differentially infect pathogenic strains of E. coli (i.e. E. coli O157:H7) while leaving the healthy commensal E.coli strains alone.

Another advantage of phage therapy is that it is self-repleting. A single phage infecting a cell will produce thousands of new copies of itself that can go on to kill more bacteria. However, some point to the mechanism in which viruses kill cell, by bursting it open, presents a drawback to the treatment as it will release bacterial toxins which can lead to sepsis, a potentially deadly condition in which the immune system overreacts to dieing bacteria.

But researchers are already working up a solution to this problem. At MIT, they have developed “phagemids”: engineered phages that carry plasmids instead of a whole genome. Plasmids are short piece of circular DNA that carry a couple of genes which often code for virulence factors and/or for antibiotic resistance factors. When bacteria undergo conjugation this is generally what they share to pass around resistance genes. In a phagemid, the plasmid is encoded with genes that kill the bacteria without the cell being lysed. No lysis means those internal toxins aren’t released into the body. These scientists have already had a high degree of success treating mice for peritonitis.

Gene editing using CRISPR-Cas9 (clustered repeating interspaced short palindromic repeats) could be used to fighting bacteria. If bacteria successfully fights off infection from a phage, they save a short portion of the viruses genome in the genome. The bacteria expresses these sequences as short RNA sequences that are complementary to parts of the phage’s genome. If the bacteria becomes reinfected with the same phage, the RNA sequence binds complementary to that portion of the phages genome. Inside the cell, these RNA sequences are associated with a DNA cutting protein called Cas9, which cleave the phage genome, rendering it ineffective.

The trick with CRISPR-Cas9 is that RNA sequence can be made to be complementary to anything including bacterial resistance genes. For example, a group at Tel Aviv University has created a CRISPR-Cas9 system to target the Beta lactamase gene. This gene produces an enzyme that inactivates a wide variety of antibiotics including penicillins, cephamycins, and carbapenems. Other groups have seen excellent success targeting the genes that turn Staphylococcus aureus into MRSA.

The CRISPR sequence and the Cas9 protein will be delivered to bacteria via an engineered phage and the technique is successful at targeting both chromosomal genes and plasmid based ones. Currently, much of the research on this technique has been to re-sensitize bacteria to common antibiotics, but it is not out of the realm of possibility that CRISPR-Cas9 could be engineered to target essential bacterial genes, thus making the technique bactericidal.

The use of phages, phagemids and CRISPR-Cas9 present to us a more sophisticated and targeted way to treat infections from bacteria. However, in no way should this be thought of as a magic bullet that’s the kind of thinking that got us into trouble with antibiotics. Resistance to these techniques will happen. It is inevitable that these organisms will adapt.

Resistance to CRISPR-Cas9 in phages has already been documented in some species, so it is possible bacteria could evolve resistance to them too. Bacteria will also develop CRISPR-Cas9 systems to create a memory of the engineered phages we will use in phage therapy. But these are not reasons to abandon this technology. These should serve as a blueprint for what we should be prepared for when the day comes that resistance to our initial phage therapy options is observed. In essence, we now have the enemy’s battle plans for how they will react to our newest defense. We need to use that information to have our counter attacks waiting in the wings.


Host specificity of DNA produced by Escherichia coli : I. Host controlled modification of bacteriophage λ*

Lambda bacteriophage particles carry a “host specificity” determined by the bacterial strains on which they were produced. Upon infection of a different bacterial host (1) the phage DNA may be either accepted or rejected on the basis of this specificity, (2) if accepted, the phage multiplies and progeny phage are produced. Those progeny to which the parental phage DNA molecule is transferred, in either conserved or semi-conserved form, also receive the parental phage host specificity. All progeny containing only newly synthesized DNA receive only the specificity of the new bacterial host. It is concluded that host specificity is carried on the bacteriophage DNA.

Phage P1, present in a bacterial cell as either prophage or vegetative phage, imparts to λ DNA multiplying in the same cell a host specificity over and above that determined by the host itself. Such P1-induced specificity can be impressed equally well onto replicating and non-replicating λ DNA.

The work was supported by a grant from the Swiss National Foundation for Scientific Research.


Swan song for antibiotics? Can phage therapy and gene editing fill the gap?

It’s time to face the music: the golden age of antibiotics is over.

Humans have been in an endless war against bacteria — indeed it can kill us — and for decades antibiotics have been our only real weapon against them. They revolutionized medicine in the 20th century, and have together with vaccination led to the near eradication of many diseases in the developed world.

But despite their amazing success, and they’ve had a miracle like run, drug resistant superbugs like MRSA, CRE and VRE are slowly winning. Every year more and more people die from drug resistant infections as we inch closer back to the pre-penicillin days. Their effectiveness and easy access led to overuse, especially in live-stock raising, prompting bacteria to develop resistance. This led the World Health Organization last year to classify antimicrobial resistance as a “serious threat [that] is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country”

There is a lot of blame to be shared for this reversal: doctors prescribing antimicrobials for the common cold, patients demanding the drugs for the flu, governments de-incentivizing the development of new antibiotics and pharmaceutical companies deeming unprofitable.

Regardless of who might be blamed, this current situation was bound to happen our haphazard behavior simply hastened the arrival of this day. Whenever the goal is to kill a living organism resistance is inevitable. It’s how nature works: natural selection. In this case an antibiotic kills off all susceptible bacteria, but a hardy few resistant to the drug because of random mutations survive, and eventually thrive. On the verge of wiping out a problem, it roars back even worse than how it started.

This is how all life responds to stressors. We adapt. It shows up in the plant world as well. Look at the firestorm debate over herbicide resistant weeds in agriculture. Whether a farmer uses organic approved herbicides, conventionally bred ones (such as conventionally bred sunflower crops) or crops genetically engineered to resist glyphosate, mutant weeds survive and adapt and supersedes develop. It’s the law of nature. Yes, we can change up herbicides or create new ones, but the process then begins again. That’s true about antibiotics as well. So even if we create a new class of antibiotics, resistance will eventually follow, no matter how responsible we are with using it.

In this light, it’s clear that terms like “superbugs” and “superweeds” are misnomers because these organisms aren’t special in any way, they are simply doing exactly what nature intends them to do.

What compounds the challenge for antibiotics is that bacteria appear to be able to evolve resistance faster than any other type of organism. Conjugation (bacterial sex) allows bacteria to share resistance genes with not only members of their species, but across species and even across bacterial type (i.e. gram positive to gram negative). This is called horizontal gene transfer and allows advantageous traits to be spread among members of the current generation, instead of in future generations. Furthermore, some evidence suggests that bacteria can also sense when their back is against the wall and mutate their genome at faster rates in a last ditch effort for one of their descendants to mutate enough to develop resistance to the antibiotic.

But Darwin aside, it’s in our best interest to move beyond the era of antibiotics. Using antibiotics to treat an infection is like using a sledge hammer to pound in a nail. Sure the nail will get pounded in but there’s also going to be a large hole in your wall. Even the most narrow spectrum of antibiotics drastically affect our normal flora in ways that can have far reaching consequences on our physiology.

Phage therapy

What we need in our war against microbes are biocidal agents that are effective but also have more species specificity. One strategy in the works involves turning to the natural enemy of the bacteria, the virus for help. In other words the enemy of my enemy is my friend.

Viruses don’t just infect humans, in fact every species on the planet has numerous species of virus that can infect them. When a virus infects a bacteria, scientists have a special name for them: bacteriophage, or just phage. In bacteria, as they do in humans, viruses work by gaining access to the interior of a cell and manipulating the host’s cell machinery (i.e. enzymes, ribosomes etc) into making more copies of the virus. At a certain point, the number of copies of a virus induces the cell to burst releasing sometimes thousands of new copies of the virus, which can then go on to infect another cell and the process repeats.

Corrupting this process to fit human needs in the fight against antibiotics has actually been going on since the early days of the cold war. While the U.S. was investing in antibiotics, the Soviets were developing what is now called phage therapy. In fact in places like the country of Georgia, Russia and Poland phage therapy is still widely used today to treat infections.

One of the major benefits of phage therapy over traditional antibiotics is the specificity a phage has for its host. For the most part, a phage that infects an E. coli will not be able to infect another bacteria type, like a commensal Staphylococcus. There is even some evidence that phages are strain specific meaning we can develop phages that can differentially infect pathogenic strains of E. coli (i.e. E. coli O157:H7) while leaving the healthy commensal E.coli strains alone.

Another advantage of phage therapy is that it is self-repleting. A single phage infecting a cell will produce thousands of new copies of itself that can go on to kill more bacteria. However, some point to the mechanism in which viruses kill cell, by bursting it open, presents a drawback to the treatment as it will release bacterial toxins which can lead to sepsis, a potentially deadly condition in which the immune system overreacts to dieing bacteria.

But researchers are already working up a solution to this problem. At MIT, they have developed “phagemids”: engineered phages that carry plasmids instead of a whole genome. Plasmids are short piece of circular DNA that carry a couple of genes which often code for virulence factors and/or for antibiotic resistance factors. When bacteria undergo conjugation this is generally what they share to pass around resistance genes. In a phagemid, the plasmid is encoded with genes that kill the bacteria without the cell being lysed. No lysis means those internal toxins aren’t released into the body. These scientists have already had a high degree of success treating mice for peritonitis.

Gene editing using CRISPR-Cas9 (clustered repeating interspaced short palindromic repeats) could be used to fighting bacteria. If bacteria successfully fights off infection from a phage, they save a short portion of the viruses genome in the genome. The bacteria expresses these sequences as short RNA sequences that are complementary to parts of the phage’s genome. If the bacteria becomes reinfected with the same phage, the RNA sequence binds complementary to that portion of the phages genome. Inside the cell, these RNA sequences are associated with a DNA cutting protein called Cas9, which cleave the phage genome, rendering it ineffective.

The trick with CRISPR-Cas9 is that RNA sequence can be made to be complementary to anything including bacterial resistance genes. For example, a group at Tel Aviv University has created a CRISPR-Cas9 system to target the Beta lactamase gene. This gene produces an enzyme that inactivates a wide variety of antibiotics including penicillins, cephamycins, and carbapenems. Other groups have seen excellent success targeting the genes that turn Staphylococcus aureus into MRSA.

The CRISPR sequence and the Cas9 protein will be delivered to bacteria via an engineered phage and the technique is successful at targeting both chromosomal genes and plasmid based ones. Currently, much of the research on this technique has been to re-sensitize bacteria to common antibiotics, but it is not out of the realm of possibility that CRISPR-Cas9 could be engineered to target essential bacterial genes, thus making the technique bactericidal.

The use of phages, phagemids and CRISPR-Cas9 present to us a more sophisticated and targeted way to treat infections from bacteria. However, in no way should this be thought of as a magic bullet that’s the kind of thinking that got us into trouble with antibiotics. Resistance to these techniques will happen. It is inevitable that these organisms will adapt.

Resistance to CRISPR-Cas9 in phages has already been documented in some species, so it is possible bacteria could evolve resistance to them too. Bacteria will also develop CRISPR-Cas9 systems to create a memory of the engineered phages we will use in phage therapy. But these are not reasons to abandon this technology. These should serve as a blueprint for what we should be prepared for when the day comes that resistance to our initial phage therapy options is observed. In essence, we now have the enemy’s battle plans for how they will react to our newest defense. We need to use that information to have our counter attacks waiting in the wings.


What is BAC in biology?

A bacterial artificial chromosome (BAC) is an engineered DNA molecule used to clone DNA sequences in bacterial cells (for example, E. coli). BACs are often used in connection with DNA sequencing. Segments of an organism's DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs.

Likewise, how do you get bac? Making a BAC library To make a genomic Bacterial Artificial Chromosome (BAC) library you first have to isolate the cells containing the DNA you want to store. For animal and human BAC libraries the DNA normally comes from white blood cells. These isolated cells are then mixed with hot agarose, a jelly-like substance.

YAC stands for yeast artificial chromosome and BAC stands for bacterial artificial chromosomes. These are the vectors which are basically tools to insert the gene of interest in the host cell. The major difference between these two vectors is that of the insert size of the gene of interest.

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. The primary components of a YAC are the ARS, centromere, and telomeres from S. cerevisiae.


Note: It will be a very helpful if you have access to a lab with pipets, petri dishes, cotton swabs, and other common supplies. Talk to your teachers at school to obtain access.

  • Coliphage culture and titer determination study kit available from Ward's Science Notes: 1) This kit has enough supplies to go through the procedure one time. Purchase additional materials for repeat trials, which will help ensure your results are accurate and repeatable. This is an advanced procedure, so the details of how to repeat the procedure are not supplied. 2) This kit contains the strain E. coli B which is not a human pathogen and is safe to use in a classroom laboratory. Standard bacterial safety, detailed below, should still be followed.
    • Culture of E. coli B
    • Culture of T4r phage
    • Tryptic soy agar (125 mL)
    • 2-mL tubes of soft agar (6)
    • 9-mL dilution tubes (12)
    • 1- x 0.01-mL sterile pipets (12)
    • Sterile petri dishes (6)
    • Sterile swab
    • Pipet bulb
    • Tryptic soy agar
    • Soft soy agar
    • Tryptic soy broth
    • Petri dishes

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    'Deadman' and 'Passcode' microbial kill switches for bacterial containment

    Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.


    Influenza and Coronavirus Demise Could Lie with Phage Nanoparticles

    Given the current global viral pandemic, it wouldn’t be difficult for most to think of viruses in only a negative context. However, over the past several years, a slew of researchers and data has come to light on the benefits of using phage viruses as therapeutic options. Now, a team of investigators led by scientists at the Leibniz-Forschungsinstitut for Molecular Pharmacology (FMP) and Humboldt University (HU) has found a new use for phage in the fight against seasonal influenza and avian flu. The researchers developed a chemically modified phage capsid that “stifles” influenza viruses. Perfectly fitting binding sites cause influenza viruses to be enveloped by the phage capsids in such a way that it is practically impossible for them to infect lung cells any longer. This phenomenon has been proven in preclinical trials using human lung tissue and is being used for the immediate investigation of coronavirus infections.

    Findings from the new study were published recently in Nature Nanotechnology through an article entitled “Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry.”

    Current antiviral drugs are only partially effective because they attack viruses like influenza and coronavirus after lung cells have been infected. It would be desirable—and much more effective—to prevent infection in the first place. This is exactly what the new approach from the current study promises. The phage capsid, developed by a multidisciplinary team of researchers, envelops flu viruses so perfectly that they can no longer infect cells.

    “Preclinical trials show that we are able to render harmless both seasonal influenza viruses and avian flu viruses with our chemically modified phage shell,” explained senior study investigator Christian Hackenberger PhD, head of the department chemical biology at FMP and a professor for chemical biology at HU. “It is a major success that offers entirely new perspectives for the development of innovative antiviral drugs.”

    The new inhibitor makes use of a feature that all influenza viruses have: There are trivalent receptors on the surface of the virus, referred to as hemagglutinin protein, that attaches to sugar molecules (sialic acids) on the cell surface of lung tissue. In the case of infection, viruses hook into their victim—in this case, lung cells—like a hook-and-loop fastener. The core principle is that these interactions occur due to multiple bonds, rather than single bonds.

    It was the surface structure of flu viruses that inspired the researchers to ask the following initial question more than six years ago: Would it not be possible to develop an inhibitor that binds to trivalent receptors with a perfect fit, simulating the surface of lung tissue cells? The answer to the question lies with Q-beta phage, which has the ideal surface properties and is excellently suited to equip it with ligands—sugar molecules—as “bait.” An empty phage shell does the job perfectly.

    “We present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus,” the authors wrote. “Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo, and in vivo.”

    Lead study investigator Daniel Lauster, PhD, a former graduate student in the Group of Molecular Biophysics (HU) and now a postdoc at Freie Universität Berlin added that “Our multivalent scaffold molecule is not infectious, and comprises 180 identical proteins that are spaced out exactly as the trivalent receptors of the hemagglutinin on the surface of the virus. It, therefore, has the ideal starting conditions to deceive the influenza virus—or, to be more precise, to attach to it with a perfect spatial fit. In other words, we use a phage virus to disable the influenza virus!”

    To enable the Q-beta scaffold to fulfill the desired function, it must first be chemically modified. Produced from E. coli bacteria, the researchers used synthetic chemistry to attach sugar molecules to the defined positions of the virus shell.

    Several studies using animal models and cell cultures have proven that the suitably modified spherical structure possesses considerable bond strength and inhibiting potential. The study also enabled the research team to examine the antiviral potential of phage capsids against many current influenza virus strains, and even against avian flu viruses. Its therapeutic potential has even been proven on human lung tissue: when tissue infected with flu viruses was treated with the phage capsid, the influenza viruses were practically no longer able to reproduce.

    Additionally, high-resolution cryo-electron microscopy and cryo-electron microscopy show directly and, above all, spatially, that the inhibitor completely encapsulates the virus. Moreover, mathematical-physical models were used to simulate the interaction between influenza viruses and the phage capsid on the computer.

    “Our computer-assisted calculations show that the rationally designed inhibitor does indeed attach to the hemagglutinin, and completely envelops the influenza virus,” confirmed study co-author Susanne Liese, PhD, professor at Freie Universität Berlin. “It was therefore also possible to describe and explain the high bond strength mathematically.”

    These findings must now be followed up by more preclinical studies. It is not yet known, for example, whether the phage capsid provokes an immune response in mammals. Ideally, this response could even enhance the effect of the inhibitor. However, it could also be the case that an immune response reduces the efficacy of phage capsids in the case of repeated-dose exposure, or that flu viruses develop resistances. And, of course, it has yet to be proven that the inhibitor is also effective in human

    “Our rationally developed, three-dimensional, multivalent inhibitor points to a new direction in the development of structurally adaptable influenza virus binders. This is the first achievement of its kind in multivalency research,” Hackenberger concluded.


    What chemicals will kill P1 Phage but not E. coli? - Biology

    A major thrust of vector development has been to create vectors that will handle larger foreign DNA inserts, aiming to reduce the number of recombinants it is necessary to look at in order to identify a specific DNA sequence.

    Cosmid vectors were among the first large insert cloning vehicles developed.

    The vector replicates as a plasmid
    (it contains a ColE1 origin of replication),
    uses Amp r for positive selection and
    employs lambda phage packaging to select for recombinant plasmids carrying foreign DNA inserts 45 KB in size.

    Ligation of cosmid vector and foreign DNA fragments (SauIIIA partial digest fragments 45 kb in size) is similar to ligation into a lambda substitution vector.
    The desired ligation product is a concatemer of
    45 kb foreign DNA fragment and 5 kb cosmid vector sequences.

    This concatemer is then packaged into viral particles (remember packaging is cos site to cos site) and these are used to infect E. coli where the cosmid vector replicates using the ColE1 orignin of replication. Phage packaging serves only to select for recombinant molecules and to transfer these long DNA molecues (50 kb total) into the bacterial host (50 kb fragments transform very inefficiently while phage infection is very efficient).

    Recently, the need for prokaryotic vectors capable of replicating very large foreign DNA fragments
    (> 1 MB in size) has produced a couple of additional prokaryotic vector systems.
    One is based on another bacteriophage called P1.
    P1 phage can carry in excess of 100 KB of foreign DNA but do not seem to have found wide acceptance.

    A few years ago, a Bacterial Artificial Chromosome (BAC) vector was developed allowing foreign DNA fragements over 1 MB to be propagated in E. coli.
    These BAC vectors replicate as low copy number plasmids (to minimize the demands on the host replication system) but how do we get such large DNA fragments into E. coli?

    Standard methods for transforming E coli rely on the chemical preparation of the E coli to put them in a 'transformation competent' state.
    This typically involves incubating the bacteria in the presence of divalent cations (particularly Ca +2 ) at low temperature (4 o C) which puts the cell membrane into a 'semi-crystaline state. Addition of DNA to these 'competent cells' allow the DNA to bind to the semi-crystaline membrane. Heat shocking the cells (42 o C) remobilizes the membrane and allows the DNA to cross the membrane. This transit across the membrane is sensitive to DNA length - small plasmids cross the membrane rapidly while larger molecules are unable to cross.

    Getting very large molecules across the cell membrane relies on an alternative mechanism called electroporation. This involves mixing DNA with E coli cells and then exposing the mixture to a high-voltage pulse. The high voltage induces massive membrane rearrangements and the coincident uptake of DNA which is independant of size. BACs have been an important tool for the human genome sequencing project.

    Numerous vector systems have been developed for use in eukaryotic systems.
    The most common of these are often called 'shuttle vectors' as they replicate in both prokaryotic and eukaryotic hosts. In general, DNA manipulations and characterizations are done in prokaryotic systems, and then the manipulated DNA is reintroduced to eukaryotic systems for functional analysis.

    Yeast Vectors
    Shuttle vectors were first developed for transfering DNA fragments between E coli and S cerevisiae. These vectors contain an antibiotic resistance gene for positive selection in E coli, and E coli origin of replication (ColE1 ori), and a polylinker in the lacZ alpha-complementing fragment for insertional inactivation by the foreign DNA insert. In addition, the vector contains a yeast specific origin of replication (derived from the naturally occuring yeast plasmid called the 2 um circle - 2um ori)
    and amino-acid or N-base biosynthsis enzymes for positive selection in yeast (HIS, LEU and ADE genes)

    Mammalian Vectors
    Shuttle vectors have also been developed for use in mammalian tissue culture.
    Like the yeast shuttle vectors, mammalian shuttle vectors require sequeces enabling them to replicate in mammalian tissue culture. These eukaryotic origins of replication are typically derived from well characterized mammalian viruses - most commonly SV-40 (SV-40 ori and Large T antigen system) or Epstein-Barr virus (mononucleosis). Both systems allow the vector to replicate within the host cell as a plasmid without integrating into the genome. In addition to the origin of replication, these shuttle vectors also carry antibiotic resistance genes which function in eukaryotic cells (ie neomycin (G418) resistance, hygromycin resistance, methotrexate resistance etc).

    Finally, artificial chromosome vectors have been developed for use in yeast (YACs) and are being developed for use in mammalian cells (MACs).
    These eukaryotic vectors contain telomeric sequences (eukaryotic chromsomes are linear not circular molecules) and centromeres to ensure appropriate segregation of the artificial chromosomes as well as selectable markers. Such vector systems may gain in importance as we move towards manipulating the eukaryotic genome.


    Watch the video: M13 PHAGE VECTORS English u0026 Malayalam (June 2022).


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