I can not find some useful sources unfortunately.
Please tell me about some important characteristics of DH5 alpha.
What makes DH5 alpha suitable for the gene cloning?
There are two possible sources: One is the article from the Bethesda Research Laboratories (reference 1), which names a few, the other is the book "DNA cloning: A practical approach" (see reference 2)., which sits here in my bookshelf.
Taking both together, the DH5 alpha strain is derived from the DH5 strain with the introduction of a few additional features. The naming DH are the initials of Douglas Hanahan which developed the strain. The strain is easy to transform with high efficency.
The new features of DH5 alpha are the recA and the endA1 mutations:
- The endA1 mutation inactivates an intracellular endonuclease that degrades plasmid DNA.
- The recA mutation eliminates homologous recombination. This reduces the chance for deletions and plasmid multimerization.
The DH5 strain also has the following features:
- The hsdR17 mutation eliminates the restriction endonuclease of the EcoKI restriction-modification system, so DNA lacking the EcoKI methylation will not be degraded. DNA prepared from hsdR strains that are wt for hsdM will be methylated and can be used to transform wt E. coli K-12 strains.
- Δ(lacZ)M15 is the alpha acceptor allele needed for blue-white screening with many lacZ based vectors.
Of special interest are the references 3 and 4, which gives you all the details you want to know.
- Bethesda Research Laboratories. 1986. BRL pUC host: E. coli DH5α competent cells. Focus 8(2):9. The article is on page 13 in this paper.
- DNA Cloning: A Practical Approach. Glover, D. M. (ed.), 1985, Vol. 1, p. 109, IRL Press, McLean, Virginia
- Choosing a Competent E.coli Strain
- E. coli genotypes
Mutations: Meaning, Characteristics and Detection | Genetics
In this article we will discuss about:- 1. Meaning of Mutations 2. Characteristics of Mutations 3. Classification 4. Types 5. Agents 6. Detections 7. Nutritional Deficiency Method 8. Spontaneous Mutations 9. Applications of Mutations in Crop Improvement.
- Meaning of Mutations
- Characteristics of Mutations
- Classification of Mutations
- Types of Mutations
- Agents of Mutations
- Detections of Mutations
- Nutritional Deficiency Method of Mutations
- Spontaneous Mutations
- Applications of Mutations in Crop Improvement
1. Meaning of Mutations:
Mutation refers to sudden heritable change in the phenotype of an individual. In the molecular term, mutation is defined as the permanent and relatively rare change in the number or sequence of nucleotides. Mutation was first discovered by Wright in 1791 in male lamb which had short legs.
Later on mutation was reported by Hugo de Vries in 1900 in Oenothera, Morgan (1910) in Drosophila (white eye mutant) and several others in various organisms. The term mutation was coined by de Vries.
2. Characteristics of Mutations:
Mutations have several characteristic features.
Some of the important characteristics of mutations are briefly presented below:
i. Nature of Change:
Mutations are more or less permanent and heritable changes in the phenotype of an individual. Such changes occur due to alteration in number, kind or sequence of nucleotides of genetic material, i.e., DNA in most of the cases.
Spontaneous mutations occur at a very low frequency. However, the mutation rate can be enhanced many fold by the use of physical and chemical mutagens.
The frequency of mutation for a gene is calculated as follows:
Frequency of gene mutation = M / M + N
where, M = number of individuals expressing mutation for a gene, and
N = number of normal individuals in a population.
iii. Mutation Rate:
Mutation rate varies from gene to gene. Some genes exhibit high mutation rate than others. Such genes are known as mutable genes, e.g., white eye in Drosophila. In some genomes, some genes enhance the natural mutation rate of other genes. Such genes are termed as mutator genes.
The example of mutator gene is dotted gene in maize. In some cases, some genes decrease the frequency of spontaneous mutations of other genes in the same genome, which are referred to as anti-mutator genes. Such gene has been reported in bacteria and bacteriophages.
iv. Direction of Change:
Mutations usually occur from dominant to recessive allele or wild type to mutant allele. However, reverse mutations are also known, e.g., notch wing and bar eye in Drosophila.
Mutations are generally harmful to the organism. In other words, most of the mutations have deleterious effects. Only about 0.1% of the induced mutations are useful in crop improvement. In majority of cases, mutant alleles have pleiotropic effects. Mutations give rise to multiple alleles of a gene.
vi. Site of Mutation:
Muton which is a sub-division of gene is the site of mutation. An average gene contains 500 to 1000 mutational sites. Within a gene some sites are highly mutable than others. These are generally referred to as hot spots. Mutations may occur in any tissue of an organism, i.e., somatic or gametic.
vii. Type of Event:
Mutations are random events. They may occur in any gene (nuclear or cytoplasmic), in any cell (somatic or reproductive) and at any stage of development of an individual.
The same type of mutation may occur repeatedly or again and again in different individuals of the same population. Thus, mutations are of recurrent nature.
3. Classification of Mutations:
Mutations can be classified in various ways. A brief classification of mutations on the basis of:
(7) Visibility is presented in Table 14.1.
4. Types of Mutants:
The product of a mutation is known as mutant. It may be a genotype or an individual or a cell or a polypeptide.
There are four main classes of identifiable mutants, viz:
These are briefly described below:
Morphological mutants refer to change in form, i.e., shape, size and colour. Albino spores in Neurospora, curly wings in Drosophila, dwarf peas, short legged sheep are some examples of morphological mutants.
In this class, the new allele is recognized by its mortal or lethal effect on the organism. When the mutant allele is lethal all individuals carrying such allele will die but when it is semi-lethal or sub-vital some of the individuals will survive.
iii. Conditional Lethal:
Some alleles produce a mutant phenotype under specific environmental conditions. Such mutants are called restrictive mutants. Under other conditions they produce normal phenotype and are called permissive. Such mutants can be grown under permissive conditions and then be shifted to restrictive conditions for evaluation.
iv. Biochemical Mutant:
Some mutants are identified by the loss of a biochemical function of the cell. The cell can assume normal function, if the medium is supplemented with appropriate nutrients. For example, adenine auxotroph’s can be grown only if adenine is supplied, whereas wild type does not require adenine supplement.
Mutagens refer to physical or chemical agents which greatly enhance the frequency of mutations. Various radiations and chemicals are used as mutagens. Radiations come under physical mutagens. A brief description of various physical and chemical mutagens is presented below:
Physical mutagens include various types of radiations, viz. X-rays, gamma rays, alpha particles, beta particles, fast and thermal (slow) neutrons and ultra violet rays (Table 14.2).
A brief description of these mutagens is presented below:
X-rays were first discovered by Roentgen in 1895. The wavelengths of X-rays vary from 10 -11 to 10 -7 . They are sparsely ionizing and highly penetrating. They are generated in X-rays machines. X-rays can break chromosomes and produce all types of mutations in nucleotides, viz., addition, deletion, inversion, transposition, transitions and trans-versions.
These changes are brought out by adding oxygen to deoxyribose, removing amino or hydroxyl group and forming peroxides. X-rays were first used by Muller in 1927 for induction of mutations in Drosophila.
In plants, Stadler in 1928 first used X-rays for induction of mutations in barley. Now X-rays are commonly used for induction of mutations in various crop plants. X-rays induce mutations by forming free radicals and ions.
Gamma rays are identical to X-rays in most of the physical properties and biological effects. But gamma rays have shorter wave length than X-rays and are more penetrating than X-rays. They are generated from radioactive decay of some elements like 14C, 60C, radium etc.
Of these, cobalt 60 is commonly used for the production of Gamma rays. Gamma rays cause chromosomal and gene mutations like X-rays by ejecting electrons from the atoms of tissues through which they pass. Now a days, gamma rays are also widely used for induction of mutations in various crop plants.
iii. Alpha Particles:
Alpha rays are composed of alpha particles. They are made of two protons and two neutrons and thus have double positive charge. They are densely ionizing, but lesser penetrating than beta rays and neutrons. Alpha particles are emitted by the isotopes of heavier elements.
They have positive charge and hence they are slowed down by negative charge of tissues resulting in low penetrating power. Alpha particles lead to both ionization and excitation resulting in chromosomal mutations.
iv. Beta Particles:
Beta rays are composed of beta particles. They are sparsely ionizing but more penetrating than alpha rays. Beta particles are generated from radioactive decay of heavier elements such as 3H, 32P, 35S etc. They are negatively charged, therefore, their action is reduced by positive charge of tissues. Beta particles also act by way of ionization and excitation like alpha particles and result in both chromosomal and gene mutations.
v. Fast and Thermal Neutrons:
These are densely ionizing and highly penetrating particles. Since they are electrically neutral particles, their action is not slowed down by charged (negative or positive) particles of tissues. They are generated from radioactive decay of heavier elements in atomic reactors or cyclotrons. Because of high velocity, these particles are called as fast neutrons.
Their velocity can be reduced by the use of graphite or heavy water to produce slow neutrons or thermal neutrons. Fast and thermal neutrons result in both chromosomal breakage and gene mutation. Since they are heavy particles, they move in straight line. Fast and thermal neutrons are effectively used for induction of mutations especially in asexually reproducing crop species.
vi. Ultraviolet Rays:
UV rays are non-ionizing radiations, which are produced from mercury vapour lamps or tubes. They are also present in solar radiation. UV rays can penetrate one or two cell layers. Because of low penetrating capacity, they are commonly used for radiation of micro-organisms like bacteria and viruses.
In higher organisms, their use is generally limited to irradiation of pollen in plants and eggs in Drosophila UV rays can also break chromosomes. They have two main chemical effects on pyrimidine’s.
The first effect is the addition of a water molecule which weakens the H bonding with its purine complement and permits localized separation of DNA strands. The second effect is to join pyrimidines to make a pyrimidine dimer.
This dimerization can produce TT, CC, UU and mixed pyrimidine dimers like CT. Dimerization interferes with DNA and RNA synthesis. Inter-strand dimers cross link nucleic acid chains, inhibiting strand separation and distribution.
There is a long list of chemicals which are used as mutagens. Detailed treatment of such chemicals is beyond the scope of this discussion.
The chemical mutagens can be divided into four groups, viz:
A brief description of some commonly used chemicals of these groups is presented below.
a. Alkylating Agents:
This is the most powerful group of mutagens. They induce mutations especially transitions and transversions by adding an alkyl group (either ethyl or methyl) at various positions in DNA. Alkylation produces mutation by changing hydrogen bonding in various ways.
The alkylating agents include ethyl methane sulphonate (EMS), methyl methane sulphonate (MMS), ethylene imines (EI), sulphur mustard, nitrogen mustard, etc.
Out of these, the first three are in common use. Since the effect of alkylating agents resembles those of ionizing radiations, they are also known as radiomimetic chemicals. Alkylating agents can cause various large and small deformations of base structure resulting in base pair transitions and transversions.
Transversions can occur either because a purine has been so reduced in size that it can accept another purine for its complement, or because a pyrimidine has been so increased in size that it can accept another pyrimidine for its complement. In both cases, diameter of the mutant base pair is close to that of a normal base pair.
b. Base Analogues:
Base analogues refer to chemical compounds which are very similar to DNA bases. Such chemicals sometimes are incorporated in DNA in place of normal base during replication. Thus, they can cause mutation by wrong base pairing. An incorrect base pairing results in transitions or transversions after DNA replication. The most commonly used base analogues are 5 bromo uracil (5BU) and 2 amino purine (2AP).
5 bromo uracil is similar to thymine, but it has bromine at the C5 position, whereas thymine has CH3 group at C5 position. The presence of bromine in 5BU enhances its tautomeric shift from keto form to the enol form. The keto form is a usual and more stable form, while enol form is a rare and less stable or short lived form. Tautomeric change takes place in all the four DNA bases, but at a very low frequency.
The change or shift of hydrogen atoms from one position to another either in a purine or in a pyrimidine base is known as tautomeric shift and such process is known as tautomerization.
The base which is produced as a result of tautomerization is known as tautomeric form or tautomer. As a result of tautomerization, the amino group (-NH2) of cytosine and adenine is converted into imino group (-NH). Similarly keto group (C = 0) of thymine and guanine is changed to enol group (-OH).
5BU is similar to thymine, therefore, it pairs with adenine (in place of thymine). A tautomer of 5BU will pair with guanine rather than with adenine. Since the tautomeric form is short-lived, it will change to keto form at the time of DNA replication which will pair with adenine in place of guanine.
In this way it results in AT GC and GC —> AT transitions. The mutagen 2AP acts in a similar way and causes AT <-> GC transitions. This is an analogue of adenine.
c. Acridine Dyes:
Acridine dyes are very effective mutagens. Acridine dyes include, pro-flavin, acridine orange, acridine yellow, acriflavin and ethidium bromide. Out of these, pro-flavin and acriflavin are in common use for induction of mutation. Acridine dyes get inserted between two base pairs of DNA and lead to addition or deletion of single or few base pairs when DNA replicates (Fig. 14.1).
Thus, they cause frameshift mutations and for this reason acridine dyes are also known as frameshift mutagens. Proflavin is generally used for induction of mutation in bacteriophages and acriflavin in bacteria and higher organisms.
d. Other Mutagens:
Other important chemical mutagens are nitrous acid and hydroxy amine. Their role in induction of mutation is briefly described here. Nitrous acid is a powerful mutagen which reacts with C6 amino groups of cytosine and adenine. It replaces the amino group with oxygen (+ to – H bond). As a result, cytosine acts like thymine and adenine like guanine.
Thus, transversions from GC —> AT and AT —> GC are induced. Hydroxylamine is a very useful mutagen because it appears to be very specific and produces only one kind of change, namely, the GC —> AT transition. All the chemical mutagens except base analogues are known as DNA modifiers.
6. Detection of Mutation:
Detection of mutations depends on their types. Morphological mutations are detected either by change in the phenotype of an individual or by change in the segregation ratio in a cross between normal (with marker) and irradiated individuals. The molecular mutations are detected by a change in the nucleotide, and a biochemical mutation can be detected by alteration in a biochemical reaction.
The methods of detection of morphological mutants have been developed mainly with Drosophila. Four methods, viz., (1) CIB method, (2) Muller’s 5 method, (3) attached X-chromosome method, and (4) curly lobe plum method are in common use for detection of mutations in Drosophila.
A brief description of each method is presented below:
This method was developed by Muller for detection of induced sex linked recessive lethal mutations in Drosophila male. In this technique, C represents a paracentric inversion in large part of X-chromosome which suppresses crossing over in the inverted portion. The I is a recessive lethal. Females with lethal gene can survive only in heterozygous condition.
The B stands for bar eye which acts as a marker and helps in identification of flies. The I and B are inherited together because C does not allow crossing over to occur between them. The males with CIB chromosome do not survive because of lethal effect.
The important steps of this method are as follows:
(a) A cross is made between CIB female and mutagen treated male. In F1 half of the males having normal X-chromosome will survive and those carrying CIB chromosome will die. Among the females, half have CIB chromosome and half normal chromosome (Fig. 14.2). From F1, females with CIB chromosome and male with normal chromosome are selected for further crossing.
(b) Now a cross is made between CIB female and normal male. This time the CIB female has one CIB chromosome and one mutagen treated chromosome received from the male in earlier cross.
This will produce two types of females, viz., half with CIB chromosome and half with mutagen treated chromosome (with normal phenotype). Both the progeny will survive. In case of males, half with CIB will die and other half have mutagen treated chromosome.
If a lethal mutation was induced in mutagen treated X-chromosome, the remaining half males will also die, resulting in absence of male progeny in the above cross. Absence of male progeny in F2 confirms the induction of sex linked recessive lethal mutation in the mutagen treated Drosophila male.
ii. Muller 5 Method:
This method was also developed by Muller to detect sex linked mutation in Drosophila. This method is an improved version of CIB method. This method differs from CIB method in two important aspects. First, this method utilizes apricot recessive gene in place of recessive lethal in CIB method. Second, the female is homozygous for bar apricot genes, whereas it is heterozygous for IB genes in CIB method.
In this method, the mutation is detected by the absence of wild males in F2 progeny. This method consists of following important steps (Fig. 14.3).
a. A homozygous bar apricot female is crossed with mutagen treated male. In F1 we get two types of progeny, viz., heterozygous bar females and bar apricot (Muller) males.
b. These F1 are inter-mated. This produces four types of individuals. Half of the females are homozygous bar apricot, and half are bar heterozygous. Among the males, half are bar apricot (Muller 5) and half should be normal. If a lethal mutation is induced, the normal male will be absent in the progeny.
iii. Attached X-Method:
This method is used to detect sex linked visible mutations in Drosophila. In this method a female in which two X-chromosomes are united or attached together is used to study the mutation (Fig. 14.4). Therefore, this method is known as attached X-method. The attached X females (XXY) are crossed to mutagen treated male. This cross gives rise to super females (XX-X), attached female (XXY), mutant male (XY) and YY.
The YY individuals die and super female also usually dies. The surviving male has received X-chromosome from mutagen treated male and Y chromosome from attached X-female. Since Y chromosome does not have corresponding allele of X-chromosome, even recessive mutation will express in such male which can be easily detected.
iv. Curly Lobe-Plum Method:
This method is used for detection of mutation in autosomes. In this method curly refers to curly wings, lobe to lobed eye and plum to plum or brownish eye. All these three genes are recessive lethal. Curly (CY) and lobed (L) genes are located in one chromosome and plum (Pm) in another but homologous chromosome.
Crossing over between these chromosomes cannot occur due to presence of inversion. Moreover, homozygous individuals for CYL or Pm cannot survive because of lethal effect. Only heterozygotes survive. Thus, this system is also known as balanced lethal system. This method consists of following steps (Fig. 14.5).
a. A cross is made between curly lobe plum (CYL/Pm) female and mutagen treated male. This produces 50% progeny as curly lobe and 50% as plum.
b. In the second generation cross is made between curly lobe female and curly lobe plum male. This will give rise to curly lobe plum, curly lobe and plum individuals in 1 : 1 : 1 ratio and homozygous curly will die due to lethal effect. From this progeny, curly lobe females and males are selected for further mating.
c. In third generation, a cross is made between curly lobe female carrying one mutagen treated autosome and curly lobe male also carrying treated autosome. This results in production of 50% progeny as curly lobe, 25% homozygous curly lobe which die and 25% progeny homozygous for treated autosomes.
This will express as autosomal recessive mutation and constitute one third of the surviving progeny. A comparison of different methods of detection of mutation in Drosophila is given in Table 14.4.
Detection of Mutations in Plants:
As stated earlier, the techniques of detection of induced mutations have been mostly developed on Drosophila. In plants, such techniques have not been developed properly. In plants, two methods are used for detection of mutations depending upon the visibility of mutations.
These methods are briefly described below:
i. Detection of Visible Mutations:
Visible mutations generally occur in qualitative or oligogenic characters. Such mutations are detected on the basis of altered phenotype.
This technique consists of following steps:
a. The seeds are treated with a mutagen. For this purpose an improved variety or strain is used.
b. The treated seeds are grown in the experimental field. These plants are known as M1 plants or M1 generation. These M1 plants are selfed to avoid outcrossing. The seeds obtained from M1 plants represent M2 generation of seed.
c. The seeds obtained from M1 plants are grown to obtain M2 plants. A sufficiently large population should be raised in M2 generation to obtain mutant phenotypes which generally occur at a low frequency.
d. A search is made to identify or to detect plants which differ from the parent variety. Such plants are isolated and their frequency is estimated. Such mutations are called macro- mutations.
In maize, a different procedure is used for detection of visible mutations. In maize, some stocks are homozygous for several recessive genes and other stocks are homozygous for several dominant genes. The seeds of homozygous dominant lines are treated with a mutagen and M1 plants are raised. These M1 plants are crossed with homozygous recessive stock.
The mutagen treated plants are used as females due to presence of some degree of male sterility in these plants as a consequence of mutagenic effect. The F1 progeny of such cross is grown and a search is made to detect plants with recessive phenotype for a specific gene. Presence of plants with recessive phenotype for a gene confirms induction of mutation.
ii. Detection of Invisible Mutation:
Invisible mutations usually occur in quantitative or polygenic characters like yield and protein content. Detection of such mutations requires quantitative measurement of such characters. For yields, the mutagen treated and untreated variety is grown in replicated trials.
If the yield of treated and untreated treatments differs significantly, the presence of mutation is indicated. Similarly, if the protein content of treated material differs significantly from the parent variety, it indicates that mutation has taken place. Such mutations are called as micro-mutations.
7. Nutritional Deficiency Method of Mutations:
This method of detection of induced mutations is used in micro-organisms like Neurospora. The normal strain is treated with a mutagen and then cultured on minimal medium. A minimal medium contains sugar, salt, inorganic acids, nitrogen and vitamin biotin. The normal strain of Neurospora grows well on the minimal medium, but a biochemical mutant fails to grow on such medium.
This confirms induction of mutation. Then minimal medium is supplemented with certain vitamins or amino acids, one by one and the growth is observed. The medium which results in normal growth of mutagen treated mould indicates that the mutant lacks synthesis of that particular vitamin or amino acid, addition of which to the minimal culture medium has resulted in normal growth of treated strain.
8. Spontaneous Mutations:
Naturally occurring mutations are known as spontaneous mutations. Such mutations are induced by chemical mutagens or radiations which are present in the external environment to which an organism is exposed. Temperature also affects the frequency of spontaneous mutations. A rise of 10°C in the temperature leads to fivefold increase in mutation rate in an organism exposed to such variation in temperature.
Drastic change of temperature in any direction produces still greater effect on mutation frequency. External environmental conditions of any type, i.e., either extremely high or low leads to increase in the mutation frequency.
Internal environment of an organism also plays an important role in the induction of spontaneous mutations. For example, spontaneous rearrangements of DNA bases result in base pair transitions. Similarly, errors in DNA repair or replication can cause spontaneous mutations.
9. Applications of Mutations in Crop Improvement:
Induced mutations are useful in crop improvement in five principal ways, viz:
(1) Development of improved varieties,
(2) Induction of male sterility,
(4) Creation of genetic variability, and
(5) Overcoming self-incompatibility.
These are briefly discussed below:
i. Development of Improved Varieties:
More than 2000 improved varieties (some directly and some by use of mutants in hybridization) have been developed through induced mutations in various field crops all over the world.
In India, induced mutations have been instrumental in developing improved varieties in wheat (NP 836, Sarbati Sonor’a, Pusa Lerma), barley (RDB 1), rice (Jagannath, IIT 48, NT 60), tomato, castor bean (Aruna, Sobhagya), cotton (MCU 7, MCU 10, Indore 2), groundnut (TGI), sugarcane (Co 8152, 8153) and several other crops.
Besides high yield, varieties have been developed with better quality, earliness, dwarfness, disease resistance and low toxin contents in various crops.
Improvement in quality has been achieved for protein content in wheat and rice, oil content in mustard and sugar content in sugarcane. Earliness has been achieved in castor (from 270 days to 140 days), rice and soybean. Dwarf varieties have been developed through the use of mutant parents in wheat, rice, Sorghum and pearl millet.
Disease resistance has been induced in oats to Victoria blight and crown rust in wheat for strip rust in barley for mildew in groundnut for leaf spot and stem rust in sugarcane for red rot in apple for mildew, etc. Low toxin content varieties have been developed in rapeseed and mustard for erusic acid and in Lathyrus sativa for neurotoxin content.
ii. Induction of Male Sterility:
Induced mutations have been useful in induction of male sterility in some crop plants. Genetic male sterility has been induced in durum wheat using radiations. CMS mutants have been induced in barley, sugarbeet, pearl millet and cotton. Use of GMS and CMS lines helps in reducing the cost of hybrid seed production.
iii. Production of Haploids:
Use of X-ray irradiated pollens has helped in production of haploids in many crops. Chromosome doubling of these haploids results in the development of inbred lines which can be utilized in the development of commercial hybrids.
iv. Creation of Genetic Variability:
Induced mutations are very effective in creating genetic variability for various economic characters in crop plants. Induced mutations have been used for increasing the range of genetic variability in barley, oats, wheat and many other crops. In asexually propagated crops like sugarcane and potato, somatic mutations may be useful, because the mutant plant can be multiplied as a clone.
v. Overcoming Self-Incompatibility:
Mutation of S gene by irradiation offers a solution to the production of self-fertile plants in self-incompatible species. This has been successful in case of Prunusovium. Besides this practical application in crop improvement, induced mutations are of fundamental interest in genetical studies.
Induced mutations have some limitations also. Most of the mutations are deleterious and undesirable. Identification of micro-mutations, which are more useful to a plant breeder is usually very difficult. Since mutations are produced at a very low frequency, a very large plant population has to be screened to identify and isolate desirable mutants.
Protein Expression and Purification Core Facility
Transformation is the process of getting the recombinant vector from a reaction mixture or vector solution into E. coli cells. To enable the cells to take up circular vector DNA they have to be made competent. The method for the preparation of competent cells depends on the transformation method used and transformation efficiency required.
- For a high transformation efficiency, we use electroporation and electroporation-competent cells. Here is a protocol for preparing electrocompetent E. coli.
- If a lower efficiency is sufficient, we use heat shock transformation and chemically competent cells. Here is a protocol for preparing heat shock competent E. coli
The choice of the E. coli host strain depends on the goal of the transformation.
- The transformation of a ligation mix should be done in a recA- cloning strain, such as DH5a, NovaBlue or XL1-Blue. Always use a negative control with only vector DNA.
- Depending on the background of non-recombinants (from a ligation mix containing only digested vector), a number of transformants (3-12) should be picked and checked for the presence of the right insert by restriction analysis or colony PCR.
Note: We recommend colony PCR testing prior to DNA Minipreps as you can get the result for positive clones while the cultures inoculated with the same colonies are incubating. You prepare DNA only from
1-2 of the PCR-positive clones instead of preparing from all inoculated colonies and use restriction digestion for testing. It will save you time and money for restriction enzyme and Miniprep columns, additionally you can screen more colonies in parallel.
(Should your PCR not work for some reason, at least you still have your colonies growing, therefore you don't lose anything while waiting for them to grow.)
Ag43 TEV Release System
The PRISMO release system works by utilizing the Ag43 autotransporter and the TEV protease. The translated SCI peptides are directed towards the periplasmic membrane via the pelB signal sequence. The SCI peptides are fused with a TEV protease cut site and are displayed on the surface of the cell via Ag43 autotransporters continuously (with a constitutive promoter). Along with the SCI analogs TEV proteases are also displayed on the surface of the cell when induced with arabinose, again via the Ag43 autotransporter. Due to the fluid mosaic model of the cell membrane these autotransporters move and collide with each other in the cell membrane. If these collisions are in the right position and angle the TEV proteases cleave the SCI analogs from the autoransporter at the fused TEV protease site releasing them into the medium. To test the release system we created a construct which has sfGFP instead of the SCI. We were then able to quantify the protein release via a M5 spectrophotometer . .
The high impact of an animal disease event associated with the accidental release of high consequence animal disease viruses necessitates the precaution of having procedures to ensure the complete inactivation of viruses and + ssRNA viral genomes. In the studies presented here, boiling alone for five minutes was demonstrated to inactivate African horse sickness virus, African swine fever virus, foot-and-mouth disease virus, lumpy skin disease virus, porcine parvovirus, swine vesicular disease virus, vesicular exanthema of swine virus, classical swine fever virus, and vesicular stomatitis virus (Tables 4 and 5), and alkaline and heat treated samples spiked with FMDV and CSFV likewise demonstrated loss of virus infectivity in vitro (Table 4). The presented procedure relies not only on hot alkaline conditions (0.25 N NaOH and 65 °C for 1 h) for the degradation of all RNA, presumably to free ribonucleotides with 5’-hydroxyls and 3’-phosphates, but also on the additional step of boiling at 100 °C for 10 min after pH has been neutralized to between pH 7 and 8. As even a few breaks in viral genomic RNA are likely to prevent reconstitution of infectious virus, the standard of complete loss of rRT-PCR detectable SCR may appear to be an overly stringent assessment for verification of the DNA safety treatment procedure. Admittedly, the risk of animal infection or accidental reconstitution and release of a virus from RNA contaminants of purified DNA is extremely low. Published examples of + ssRNA viruses reconstituted in animals are few and typically involve direct injection of highly purified and abundant in vitro transcribed viral RNA within a cellular transfection medium such as lipofectamine [19, 20]. In vitro, also using cell transfection methods, recovery of virus from RNA is more common though, RNA needs to be of high quality, presumably with few or no breaks within protein coding or regulatory elements [19, 21, 22]. It is hoped that the stringent measure of RNA degradation employed herein will add confidence in the safety of exported DNA from laboratories that work with highly infectious animal disease viruses of national and international economic impact such as FMDV.
It is also hoped that the alkaline and heat treatment procedure will improve the quality of DNA over other methods of DNA safety treatment such as RNAse A treatment . In this regard, NaOH and heat treatment are expected to provide more effective degradation of protein and/or lipid encapsulated RNA as well as RNA within an RNA:RNA or RNA:DNA duplex. Indeed, similar treatments with NaOH and heat have been found to be effective in removal of RNA from cDNA preparations used in transcriptome analyses using quantitative RT-PCR or microarrays [10, 11].
Mario Alberto Flores-Valdez · Jun 30, 2017 · Academic Editor
I just encourage you to take a look at the comments from reviewer 2 and further edit your manuscript to address those comments (which can be done while in production).
Reviewer 1 · Jun 29, 2017
A substantial improvements to the new manuscript was done
A substantial improvements to method sections was implemented by authors
Validity of the findings
With the substantial modifications the manuscript is now ready to be accepted
Comments for the author
With the changes suggested and implemented the manuscript was improved
Reviewer 2 · Jun 22, 2017
The manuscript by Zhaohui Wei et al reports a new strain of Streptomyces flavogriseus. The fact that this strain is able to produce both actinomycin D and holomycin makes it unique among the hitherto known strains of S. flavogriseus. Moreover, the amount of actinomycin D produced by this strain makes it a good candidate for a future production of the drug.
The data shown in the paper is consistent and supports the conclusions. The article structure is correct.
With the modifications added, I think the article is suitable for publication.
I only have minor comments:
* I would move sentence in line 10 “The antimicrobial….diffusion method” before “Holomycin exhibited…” in line 14
*I would move “The cell viability….MTT assay” from line 12 to line 16 before “Holomycin exhibited…carcinoma cells”
- Materials and Methods:
*line 66: “… was suspend” should read “was suspended”
* I have a few concerns about some bibliography introduced. For example: HepG2 cells reference should be Knowles et al., 1980 instead of an article published last year. This is just one example, but I am afraid there are a few more of the newly introduced references. The same for DH5alpha, etc…
* Line 189: “…in three crude extracts…”. Please, mention the name of the 3 media.
* Antimicrobial activity assay: The authors should state that the the purified actinomycin D performs as the commercial one in terms of antimicrobial activity. This would strengthen their results.
* Material and methods of the new supplementary figures should be added.
Validity of the findings
The authors have addressed all the questions the reviewer had properly.
Comments for the author
The study is interesting and provides useful information. I think the manuscript has improved considerably.
- Check all containers for leakage or breakage.
- Remove the frozen cells from the dry ice packaging and immediately place the cells at a temperature below -130°C, preferably in liquid nitrogen vapor, until ready for use.
- 10% fetal bovine serum (FBS ATCC 30-2020)
- 10 µg/ml insulin
- 5.5 µg/ml transferrin
- 5 ng/ml selenium
- 40 ng/ml dexamethasone
This medium is formulated for use with a 5% CO2 in air atmosphere. Temperature
Handling procedure To insure the highest level of viability, thaw the vial and initiate the culture as soon as possible upon receipt. If upon arrival, continued storage of the frozen culture is necessary, it should be stored in liquid nitrogen vapor phase and not at -70°C. Storage at -70°C will result in loss of viability.
- Thaw the vial by gentle agitation in a 37°C water bath. To reduce the possibility of contamination, keep the O-ring and cap out of the water. Thawing should be rapid (approximately 2 minutes).
- Remove the vial from the water bath as soon as the contents are thawed, and decontaminate by dipping in or spraying with 70% ethanol. All of the operations from this point on should be carried out under strict aseptic conditions.
- Transfer the vial contents to a centrifuge tube containing 9.0 mL complete culture medium and spin at approximately 125 x g for 5 to 7 minutes.
- Resuspend cell pellet with the recommended complete medium (see the specific batch information for the culture recommended dilution ratio) and dispense into a 25 cm 2 or a 75 cm 2 culture flask. It is important to avoid excessive alkalinity of the medium during recovery of the cells. It is suggested that, prior to the addition of the vial contents, the culture vessel containing the complete growth medium be placed into the incubator for at least 15 minutes to allow the medium to reach its normal pH (7.0 to 7.6).
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- Remove and discard culture medium.
- Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
- Add 2.0 to 3.0 ml of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
- Add 6.0 to 8.0 ml of complete growth medium and aspirate cells by gently pipetting.
- Add appropriate aliquots of the cell suspension to new culture vessels.
- Incubate cultures at 37°C.
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Most of the hormones produced by the anterior pituitary perform what function?
A. regulate growth
B. regulate the sleep cycle
C. regulate production of other hormones
D. regulate blood volume and blood pressure
What is the function of the hormone erythropoietin?
A. stimulates production of red blood cells
B. stimulates muscle growth
C. causes the fight-or-flight response
D. causes testosterone production
Which endocrine glands are associated with the kidneys?
A. thyroid glands
B. pituitary glands
C. adrenal glands
Miniaturized microbial fuel cell (mMFC)
Traditional microbial fuel cells are bulky and inefficient. They are costly and less likely to be used in the industrial scale. We are fully aware of this issue, so we came up with the idea of miniaturized microbial fuel cell (mMFC).
According to our literature review, interests toward designs of the mMFC have been increasing over the last decades. Various models have been built and proved to be very successful [1, 2, 3]. For example, Shogo Inoue and colleagues have demonstrated that MFC can be as small as the size of one cent US coin (Figure 2) . Even though the volume of their MFCs is smaller than 50 uL, the current produced is increased considerably that up to 12 mW/100〖mm〗^3 was recorded regardless of modifications on both the MFCs and the bacteria. In Figure 2, four MFC designs which are different by building materials and chamber volumes were compared . There is no doubt that mMFCs can perform better. They produced about 300 times higher power densities than that of the bigger models.
Recently we came across the Bielefeld team who share the same interest and passion about MFC as a potential source of green energy. We get in touch and know that they also work on the mtrCAB complex, so we immediately see this as an opportunity for collaboration. We ask them to help us build the miniaturized MFC (Figure3) as they have facilities and materials for construction. We thank them for their generosity and wish them success in their iGEM project.
The MFC that they have sent us has 2 chambers, which in turn are divided by the cathode and the anode. Each chamber is 2.3 cm long, 2.3 cm wide and 1.5 cm high. The volume of each chamber is 7.9 cm3. The volume of the whole MFC is 15.8 cm3.
Full Microbial Fuel Cell Assembly instructions by Bielefeld here
Fortunately, our university allowed us to enter the lab this year from June to October despite the COVID-19 pandemic. TU Delft actively kept track of the news and followed instructions from health authorities, the National Institute for Public Health and the Environment (RIVM). We followed these instructions carefully.
- The team followed strict safety measures to prevent spreading of the COVID-19 virus. We were vigilant with maintaining personal hygiene by frequently and properly washing our hands, coughing and sneezing into our elbow, and using paper handkerchiefs.
- In accordance with the RIVM (Dutch National Institute for Public Health and the Environment) guidelines, we did not go to university if we had COVID-19 related symptoms or if we had been in contact with someone who tested positive.
- The TU Delft has taken several additional cleaning measures, including but not limited to, frequent cleaning of workplaces, toilets, meeting rooms and coffee machines. They also provided us with cleaning supplies such as 70% Ethanol and hand gel to make our work as safe as possible.
- In order to keep the occupancy of our buildings and laboratories as low as possible, all student projects have been carried out off-campus. We were permitted to work in the lab with a maximum of 4 people, maintaining a distance of 1.5m at all times.
- During the summer months, we were allowed to use a large lecture hall as an ‘office’, although working from home remained the norm. Throughout the project we always made a serious effort to keep a safe distance, while at the same time trying to do our work in the best possible way
The wetlab part of our project was performed in the ML-1 area, which is situated at the Bionanoscience department of our Faculty of Applied Sciences on the Delft University of Technology campus. ML-1 is considered to be the lowest biosafety level, corresponding to BSL-1. After passing the mandatory ML1 tests we were allowed to enter and work at two labs.We worked in one regular ML-1 lab and one ML-1 lab meant for bacteriophage work only (Figure 1).
Prior to starting our experiments, all (wetlab) team members passed the obligatory safety tests about:
- General safety of the Faculty of Applied Sciences
- General Laboratory safety
- Biological safety ML-1
- Laser safety for Laser Users
Below you can find a summary of the rules employees and students at the TU Delft have to follow to guarantee a safe work environment:
- In case of emergency or a minor accident we must call the internal alarm number. We must explain who we are, where we are and what the emergency is. The emergency centre of TU Delft will send the in-house emergency response team (Bedrijfshulpverlening, BHV). If needed, the emergency centre of TU Delft will contact the external emergency services.
- In case that the alarm goes off we should first think of our own safety. We must secure our workstation and leave the building as soon as possible, taking the nearest (emergency) exit. If possible, we must warn colleagues and assist people with disabilities. We have to go to the nearest assembly point/checkpoint. At the assembly point/checkpoint, we have to wait for instructions of the in-house emergency response team (Bedrijfshulpverlening, BHV).
- When we see gas, smoke or fire we must first think of our own safety. We have to press the red fire alarm button. If we feel confident we can extinguish small fires. If possible, we must warn colleagues and assist people with disabilities. We have to go to the nearest assembly point/checkpoint. At the assembly point/checkpoint, wait for instructions of the in-house emergency response team (Bedrijfshulpverlening, BHV).
- We must always report incidents, accidents, near misses and unsafe situations to the Health, Safety and Environmental advisor of the faculty.
In addition to completing the safety tests, we also received lab training on how to safely operate most of the techniques used in basic experiments. We agreed upon rules on how to work safely in our lab to minimize potential risks to laboratory personnel and the environment. We also learned how to discard different types of waste and how to minimize contamination risk. To work in the special bacteriophage lab we received a separate training, as extra safety measures are required to contain the phages and prevent contamination. We learned how to clean phage contaminated surfaces properly and how to correctly perform experiments with phages.
Apart from working safely we also made sure we designed experiments that we could perform safely. We wrote a safety report to evaluate the risks of our experiments including: experimental details, designs, safety regarding COVID-19 and biological safety information. This safety report was approved by Susanne Hage, wetlab coordinator of the department of Bionanoscience of the TU Delft, and Marinka Almering, Biosafety Officer of the Faculty of Applied Sciences of TU Delft. When we made adjustments to our project, we updated the proposal and got it approved before starting the new experiments.
Safe Experimental Design
We designed all our experiments to comply with the ML-1 safety level. Classification of microorganisms or toxins can differ between countries or regions, we followed the legislation of the TU Delft. Prior to working in the lab we checked that all organisms, vectors and parts are in accordance with the ML-1 level and the Dutch law.
To comply with our ML-1 lab we made sure that all our microorganisms were ML-1. We used E. coli BL21 (DE3) and E. coli DH5 alpha as our chassis organisms. In addition we used E. coli BL21 (DE3) as a host for our phage engineering experiments. We decided to work with the T7 bacteriophage, as it is also classified as ML-1. We exclusively performed phage experiments in a separate bacteriophage lab to prevent unintended E. coli infection (Figure 1).
We made sure to express safe inserts. The Cry7Ca1 toxin is specific to insects, and can be used in an ML-1 lab without separate acceptance from the GMO bureau.
We also expressed YmdB (RNAse II inhibitor), Mini-III (cleaves shRNA), 4-hydroxybenzoate Decarboxylase (degrades phenol) (for more information see Design). These molecules were all not toxic and classified as ML-1. All of these molecules have not been reported to be harmful to humans.
For our research we used different backbones vectors: pKD46, G322 - pUC57 - OriLR - deGFP, pTWIST_bsdBCCD_CO, pSB1C3_BPUL_GA, pTWIST_Cry, pBbB7a - GFP, pBbA2k - RFP, pBbE8c - RFP, pCas9-CR4, pKDsgRNA-p15. Some of these plasmids were kindly provided by other labs from the BN department and some were ordered via Addgene. Material transfer agreement (MTA) regulations were followed according to the rules.
We used several chemicals that are hazardous, these include Ethidium bromide, 2X Laemmli Sample Buffer and SYBR™ Safe. We have only worked with these chemicals in low concentrations and solely worked with these toxins in the designated area. This area was marked with special tape, and we agreed to all work with gloves in that area these gloves were colored differently compared with the general stock. In addition, any contaminated equipment did not leave this area.
Safety is one of our important design requirements. PHOCUS is a targeted bacteriophage-based biopesticide used to control the desert locust crisis. Our aim is to kill the locusts by producing toxic molecules in their gut. These molecules will be produced by the gut bacteria after infection with our engineered bacteriophage. The toxins we want to produce are the locust specific Cry7Ca1 toxin and short-hairpin RNAs (shRNAs) that target essential locust genes (for more information see Design).
Whilst working on our project, we learned that in biology, nothing is 100% safe. Therefore, we decided to focus on identifying potential risks and having appropriate risk management measures in place to reduce these to an acceptable degree (Figure 1).
Figure 1. Overview of Safe-By-Design measures for the design of PHOCUS.
We make use of phages for toxin delivery. These are viruses that can only infect bacteria . An identified safety concern is that our engineered bacteriophages could be dangerous to humans. However, we learned that this risk is negligible because:
- Phages are not able to infect human cells. Even though they can enter the human body, allergic reactions are not common in humans [2, 3, S. Hagens, personal interview].
- Phage stability in certain conditions varies for different phages . For our project, it is important to choose a phage that is stable at the pH of the desert locust gut, which is pH 7-8 [J. Vanden Broeck, personal interview]. A phage that is stable at this pH is not likely to survive at the pH of the human stomach (pH 1-2) .
- If phages do survive at human stomach pH, they are not likely to affect the human gut microbiota [S. Hagens, personal interview]. Nonetheless, more research on the exact effect of lytic phages on the gut microbiota has to be done [6,7].
An additional safety concern is that our phages could be dangerous to animals and/or insects. Research has looked at the influence of phages on animal microbiota, but this impact is not well understood yet . Regarding insects, research has been done on the effect of oral administration of phages by flies and bees [9, 10]. This seemed to have little effect. All in all, more research regarding the effect phages have on the microbiota of animals and insects should be done (see Field trials).
Another safety concern is the persistence of our engineered phages in nature after being released. However, the chance of our phages persisting for a long time is low as:
- Phages are unstable when exposed to persistent levels of UV [J.B. Jones, personal interview, 11]. Thus, outside the locust gut, phages will not persist long.
- Phage stability varies with temperature. The risk of persistence in nature may be reduced by choosing a phage that is predominantly stable at locust body temperatures 25-31 degrees Celsius .
To test the stability of PHOCUS inside the locust, and instability outside of it, we have worked out which data needs to be collected to be admissible for field trials (see Field trials).
Phages rely on the metabolic processes of the host cell to enable viral replication. They are known to follow two possible life cycles the lytic cycle and the lysogenic cycle . A lytic (virulent) phage does not integrate its genetic material into that of its host . While the life cycle of a lysogenic (temperate) phage consists of the viral genome, referred to as the prophage, being inserted into the genome of the host . PHOCUS will use a lytic phage, for several reasons:
- It will minimize the risks of horizontal gene transfer and/or phage mediated transduction . This could otherwise give certain bacteria an advantage, e.g. antibiotic resistance, and thereby disrupt the ecosystem . Given that our project focuses on lytic phages, generalized transduction is relevant. Generalized transduction involves the integration of random genetic information from the host chromosome into the phage. This rare event happens during phage replication with a prevalence rate of 1 in 11,000 phages, just before the viroid is enclosed inside the capsid of the phage .
- The lytic cycle ends with cell lysis and, therefore, cell death, reducing the chance of creating a new GMO.
- Due to the properties of the lytic cycle, the phages that reach their target bacteria will propagate quickly. This will lead to large increases in the amount of toxin and shRNA produced. When the host bacteria bursts, the Cry7Ca1 toxin, shRNA and RNAi associated proteins are effectively secreted, negating the issue of exporting the produced compounds out of the host. As the compounds are released in close proximity to the gut wall, they can easily reach their target. Higher Cry7Ca1 toxin and shRNA concentrations at the gut wall is correlated with a faster death rate, thereby making PHOCUS more effective.
We engineer phages to replace inessential genes with a gene coding for Cry7Ca1 toxin or shRNA. The risks involved with the spreading of engineered phages depend on the type of phage and the type of genetic modification. There are different concerns, apart from the previously mentioned horizontal gene transfer:
- Phage dissemination into the environment: Engineered phages with an expanded host range have a significantly increased change to spread into the environment . We decided upon using a phage cocktail, as there was not one single species of bacteria always present within the locust gut. With the use of this cocktail we spread multiple different phages, while these all separately have a chance to propagate. Increasing the total chance of propagation. Therefore we chose to engineer phages with a naturally narrow host range, so the propagation chance of our modified phage cocktail outside the locust is reduced.
- Production of harmful proteins: since phages can be engineered to express all types of proteins, the risks involved with the use of engineered phages strongly depend on the type of modification. We explicitly chose not to insert any sequences harmful for either the environment, humans or other animals.
- Persistence of the applied mutation in nature: To examine the stability of the mutation in engineered phages, the presence of the mutation can be determined over several generations of phages, as described by Nobrega et al. . In short, the engineered phage can be propagated in its host for several generations and the presence of the mutation can be confirmed by PCR after each generation. If our insert is extremely stable, our mutation could spread through genetic populations.
Insecticidal crystal (Cry) toxins from Bacillus thuringiensis (Bt) are being used for biological control of pests worldwide, either through spray formulations based on spore-crystal preparations, or by introducing their genes (Cry protein or an active fragment of it) into transgenic crops. [20, 21]. In our project, we focus on the Cry toxin Cry7Ca1 identified in the B. thuringiensis strain BTH-13 that has been described to be effective against locust of the species Locusta migratoria manilensis, by puncturing the gut lining [22, 23].
Regarding safety we chose to use Cry7Ca1 due to different reasons:
- The Cry toxins are highly specific to their target insects, and therefore kill a limited number of species . Cry7Ca1 has been reported to have low or no toxicity towards Lepidoptera, Coleoptera and Diptera .
- Specificity of Cry toxins is provided by the midgut environment of the insect, that promotes toxin activation from the protoxin and by the binding of the Cry toxin only to gut membranes that display the appropriate matched receptors .
- Humans do not have the same gut conditions nor the same receptors as insects. The Cry toxin should pass through us with no effect, being ingested like any other protein when eating food .
- These proteins have shown to break down in simulated digestive systems of humans . For allergen testing, we additionally tested the sequence of the Cry toxin, using the protocol from the Baltimore iGEM team of 2017, which came back as non allergenic .
Our RNA of interference (RNAi) approach relies on the small interfering RNA (siRNA) processing machinery. We chose to use the RNAi approach because:
- siRNAs needs 100% sequence complementarity to the targeted messenger RNA (mRNA) in order to cleave them , ensuring extra specificity. Specificity is very important as any eukaryotic cell possessing active RNAi machinery is susceptible to silencing with our shRNAs.
- We can ensure specificity by choosing the right short hairpin RNA (shRNA) target. As phages will be sprayed in the environment, other species might be exposed to our shRNAs. The shRNA produced has to be screened for potential off targets to prevent fortuitous gene silencing in coexisting species in or nearby locust swarms, as well as humans and animals, through the use of transcriptomic databases. When choosing our target, we must ensure choosing specific targets, in order to prevent the surfacing of unwanted phenotypes, i.e. degregarisation  or increased food intake [31, 32].
For our biopesticide to work, bacteriophages that specifically target the bacteria present in the locust gut must be chosen as a delivery vector. Although the locust gut is not dominated by a single bacterial species, the desert locust maintains a constant population of the Enterobacter genus . To ensure sufficient toxin production, the biopesticide consists of a cocktail of phages targeting a variety of bacteria from the Enterobacter genus. Unfortunately, bacteria from the Enterobacter genus are not necessarily specific to the locust gut only. Although the Enterobacter genus is not a geographically conserved core member of the human microbiome, there are certain human populations that do show the presence of Enterobacter in their microbiome . Therefore, the possibility exists that our biopesticide could infect and kill the Enterobacter bacteria of certain humans or other organisms, while simultaneously producing the locust specific toxin.
However, these risks are small because:
- In humans, it is unlikely that the unintended killing of bacterial species by phages has negative consequences for human health, due to the strong selection pressure for non-pathogenic bacteria [S. Hagens, personal interview].
- For humans, the first line of defense against phages is the low pH in the stomach as most phages are sensitive to acidic conditions. .
- The risks for other organisms are also limited. Not only humans but also other organisms that contain Enterobacter in their gut [O. Lavy, personal interview] could potentially be at risk. However, the toxins that are produced are highly specific to locusts and should not harm those organisms. Nonetheless, to ensure our biopesticide is safe for other animals, more metagenomic data studies should be performed on different gut microbiomes.
Encapsulation is a means by which the environment of a single molecule can be rigorously controlled . Several types of encapsulation exist, for example liposomes and polymeric microcapsules such as alginates .
- Encapsulation can act as a physical barrier to prevent off-target infection. The Enterobacter genus is known to prompt plant growth as they are involved in nitrogen fixation, soil phosphorus solubilisation and the production of antibiotics . A physical barrier is needed for our phages, regardless of the nature of the engineering we are performing, to avoid ecological imbalances on the vegetation the phage is sprayed upon.
A clear encapsulation method that meets our design requirements is difficult to find at first glance. Further research on encapsulation for our bacteriophages should be done.
When exposed to pesticides, locusts with different phenotypic traits have a different chances of survival. Genes resulting in a higher chance of survival are more likely to be passed onto offspring. If this occurs for longer periods, pesticide resistant locusts can become dominant in a population. We have to prevent resistance for our pesticide to remain effective. The aforementioned traits include:
The Cry7Ca1 toxin attaches to receptors in the locust gut and pokes holes in the gut lining. Some locusts may have slight genetic alterations causing changes in the Cry7Ca1 receptor, resulting in inefficient binding, or none at all. Locusts with this trait will most likely pass it onto their offspring. The probability that this happens is difficult to estimate. Nevertheless, the risk that the desert locust becomes resistant to Cry7Ca1 is enhanced by:
- High exposure of locusts to Cry7Ca1 .
- Fast reproduction, resulting in many generations [39, 40].
The risk that this occurs is decreased by:
- The risk that this occurs is decreased by:
- Low initial frequency of phenotypic traits favoring survival .
Three different scenarios in which the desert locust evolves to become resistance to RNAi exist :
- The RNAi machinery of the desert locust is impaired due to mutations . However, an imposed RNAi system decreases chances of survival in a swarm due to virus susceptibility and, therefore, is not likely that this mutation propagates through the population.
- The target sequence in the desert locust is mutated to the extent that its mRNA is not sufficiently homologous to the siRNAs from the shRNA anymore . In case this would happen, it would be easy to switch to another target sequence of an essential gene.
- The desert locust may obtain mutations that decrease the stability of the shRNA in the gut or the uptake of the shRNA in the hemolymph . No estimate can be provided on the magnitude of this risk.
Two methods are being applied to mitigate the risk of the desert locust becoming resistant to PHOCUS:
- Two different modes-of-action: A chance exists that a desert locust is resistant against one of the two methods. The chance that a desert locust is less susceptible for both Cry7Ca1 and RNAi at the same time, however, is negligible.
- Monitoring: When PHOCUS is applied, the genomes of the desert locust should be monitored to assess whether a particular genetic variation becomes more prevalent in the population . If so, a strategy, such as tactically spraying PHOCUS in particular areas or by developing a novel toxin with a different mode-of-action, should be devised.
Bacteria evolve resistance to phages using many mechanisms . If the target bacteria are resistant to PHOCUS, the toxins are not produced to terminate locusts. We have specifically used lytic phages to lyse all the target bacteria to limit resistance development. Still, we studied the chances for resistance developing and came up with two scenarios in which phage resistance could occur:
- The first scenario consists of bacteria developing resistance to PHOCUS, starting with a non-resistant bacterial population. Based on our mathematical model, which studies the development of resistant bacteria, it is clear that our bacteriophage would infect and kill the entire Enterobacter population within a couple of hours - which prevents the bacteria to evolve resistance to PHOCUS.
- The second scenario consists of the existence of a resistant strain in the locust gut. Based on the advice of Dr. Steven Hagens, we have chosen to use a cocktail of phages that target different receptors of the same bacterium to address problems regarding resistant bacteria. This ensures susceptibility for our target bacteria.
To reduce the risks related to GMO propagation in nature due to the release of the engineered bacteriophage, a strategy is to limit its spread in the environment . The amount of engineered phages in the environment will decrease naturally over time, making additional biocontainment measures uncalled for as:
- PHOCUS loses the competition with the wildtype phage for the host due to reduced fitness. Survival of PHOCUS depends on a selective advantage over the wildtype. However, the inserts do not confer a selective advantage to PHOCUS.
- The genetic inserts will be lost over time and the phage will return back to its wildtype sequence. This is due to the fact that transgenes often have a deleterious effect, a general consequence of disrupting the wildtype . We expect the relative fitness to decrease and hypothesize that this will result in the deletion of our insert, as is often the case for inserts in virus genomes .
- Bacteriophages are generally unstable when exposed to high temperatures and high doses of UV radiation. Different environmental factors can influence the stability of bacteriophages . Bacteriophages have difficulty with persisting on leaf surfaces because of inactivation by UV radiation [47, J.B. Jones, personal interview].
- The lytic nature of the engineered bacteriophage. A bacteriophage is dependent on the growth of their respective hosts to proliferate they are self-limiting [48, 49]. This means that if a large amount of phages is released, their respective hosts will be locally eradicated. Limiting the spread and proliferation capability.
During interviews with experts in the field of, among others, phage biology and toxicology, potential safety concerns were mapped out and discussed. To address these concerns, risk mitigation strategies were developed and lab experiments were designed to test the safety of PHOCUS. We realized that, for PHOCUS to be used in the real world, it first has to go through field trials. To be admissible for these large-scale tests, more safety tests have to be done. Therefore, we investigated which data had to be gathered and designed the experiments accordingly, in addition to studying the corresponding legislation.
From our interview with Dr. Cecile van der Vlugt, senior risk assessor at the Dutch National Institute for Public Health and the Environment, we learned that even though legislation might be absent or non-permissive, the best approach to overcoming noncompliant legislation is by performing a risk assessment of PHOCUS. We most likely have to comply with three types of regulations:
- GMO regulations: Before taking a GMO into the environment, one needs to make a risk assessment for other organisms in the environment, e.g. we need to prove that the phage will not propagate indefinitely in nature.
- Insecticide regulations: Every insecticide needs to pass regulations before it can be applied. Answering questions such as how specific is it? Does it persist in the environment?
- Food regulations: This will also apply to PHOCUS as it is a ‘living’ insecticide. Regulators will look at toxin/pathogenic effects of the modification made in the phage and how it could be passed to other organisms.
There are many different risks associated with the implementation of PHOCUS for which data should be gathered at a lab scale. This way we can prove the theoretical claims we have previously made, i.e. that PHOCUS is safe to humans. To move PHOCUS beyond the lab, many experiments must be performed to test its:
- Toxicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
- Pathogenicity to non-target organisms (GM phage, Cry7Ca1, RNAi)
- Stability and potential to accumulate (GM phage, Cry7Ca1, RNAi)
- Uniqueness of sequence targeted (RNAi)
- Potential gene flow of insert (GM phage)
- Specificity to locusts (CryCa1, RNAi)
It should be noted that our perception of what is acceptable keeps changing. Therefore the list should be iterated over and altered after continuous discussions with risk assessors and managers. This is extra relevant in relation to our project, as we have a novel application and therefore there may be potentially unidentified risks that could surface over time.
After completion of the lab-scale risk assessment and acceptance by the responsible authorities, PHOCUS can move to field trials. Here, PHOCUS will be subject to different tests in an outside environment, i.e. an environmental risk assessment (ERA) . The experiments performed could be the outside variant of the ones performed in the lab. This data has to be generated again as the actual environment is much more complex and could lead to different interactions. The specifics of what data exactly needs to be gathered remains imprecise. Primarily because our approach is novel and the responsible authorities have not yet drawn up the related legislation. They will likely do this using a case by case approach, as advised by scholars [52, 53]. Still, it will likely remain comparable to existing ERAs for GMO, pesticide and food regulations, e.g. the ERA for GM plants in Australia,which looks at "toxicity, allergenicity, nutritional profile, agronomic characteristics, increased disease burden, spread and persistence of the GMO, gene flow etc." . Further directives for the use of biological control agents are available . While critique of the contemporary ERA on GMOs, which use Cry toxins in transgenic plants, is also presented , it becomes clear that experts on risk assessment do not agree on the best approach. Additionally, different countries also have different procedures. Therefore, it is essential to understand that there is not one set way in which PHOCUS will gain approval in all countries, and its regulations should be assessed in close collaboration with the responsible authorities of the affected countries.
After the risk assessments have been conducted, it is important to submit these results to the Pesticide Referee Group (PRG). This is an independent group of scientific experts on pesticides. They assess the data submitted and create a shortlist of pesticides they deem acceptable. This list is specified for its intended use. The FAO advises countries on which pesticides to use. They only buy and advise pesticides that have been shortlisted by the PRG. Lastly, all countries have their own legislative freedom, therefore PHOCUS would still need to be admitted in every country individually.