Information

Plasmid choosing

Plasmid choosing


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

To design a experiment in feeding of C. elegans. It has to choose a plasmid vector to insert the gene of interest that can feed to C. elegans. Many paper are using pL4440 for the feeding vector, saying it is the first used in feeding C. elegan and because of the two T7 promotor, so it easy to perform the experiment. There also paper saying T3 promotor is not fit for feeding in C. elegans. I am curious about why T3 is not fit? and because I didn't see many paper using other vector to perform the feeding experiment in C. elegan. So I wonder that is it ok if I choose a plasmid that has two T7 promotor and is used to express in worms like pLT61, will it influence the feeding or the efficiency so that people use pL4440 for feeding in C.elegan? Or is people use pL4440 vector because it is specific designed to the feeding experiment of C. elegan? Thank you!


PREFACE: There's a text, C. elegans: Methods and Applications, specifically doi: 10.1385/1-59745-151-7:109. They outline this exact experiment in it's essence, and one of the diagrams (page 110) notes that for the purposes of RNAi in C. elegans, the T3 promoter specifically doesn't work for the bacterial feeding method, but works for injection or soaking procedures. I absolutely can't figure out why that is specifically, but i'll keep researching.

UPDATED: Much thanks to mdperry for clearing things up! As it turns out, this is a matter of experimental design. Using information from Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans, and Source Bioscience:

HT115 (DE3) is a feeding strain engineered like so,

The genotype is as follows: F-, mcrA, mcrB, IN(rrnD-rrnE)1, lambda -, rnc14::Tn10(DE3 lysogen: lavUV5 promoter -T7 polymerase) (IPTG-inducible T7 polymerase) (RNase III minus). This strain grows on LB or 2xYT plates (and is resistant to tetracycline, see below), and competent cells can be made using standard techniques.

We can go through these and look at the changes as well:

F- means no F plasmid for conjugation. mcrA/B refers to mutations made which reduce the ability of the E. coli to attack foreign DNA. IN(rrnD-rrnE)1 is an inversion in the region of some rRNA operons that I'm unsure as to the effect. Lambda- removes the lambda lysogen. You can see that they have the lavUV5 promoter for an IPTG-inducible T7 polymerase which is important for getting the E. coli to begin producing T7 polymerase to express the associated cloning vector. You can also see that HT115 (DE3) is deficient for RNase III, however, which normally degrades dsRNA, making it suitable for the feeding experiment.

The L4440 (pPD129.36) cloning vector contains two convergent T7 polymerase promoters in opposite orientation separated by a multicloning site and was made using standard cloning techniques (Timmons and Fire, 1998).

pLT61 contains 0.8 kb of unc-22 inserted into the L4440 vector.

I think the L4440 plasmid is probably easy to optimize for this experiment, and cheap. If you can access the article I linked above, they catalogue some additional plasmids. In conclusion though, it'd seem that if we in fact had a system where the E. coli themselves could produce T3 polymerase, it'd be fine to use those promoters. In the case of this experiment, the genotype of the feeding strain of bacteria nicely complements the vector being used.


Plasmids 101

Plasmids are circular pieces of DNA found in bacterial cells that replicate independently from the host’s chromosome. They’re powerful and ubiquitous tools that touch nearly all areas of biological research. In general, plasmids are used to express specific genes in target cells, introduce new elements, and can be used in a variety of other genetic manipulations. In this section of the blog, learn more about the different parts of a plasmid, molecular cloning techniques, tips for using plasmids, specific plasmid-based tools, and more.


In most cases, a high-copy plasmid vector is the best approach to produce the highest yields. For example, pBluescript has a copy number 300-500 and pUC can reach 700. You may be wondering, then, why some plasmids have a more modest value of 10-12 copy number, as is the case with pACYC. These more specialised vectors have been developed to counteract some problems caused by high-copy plasmids. Your DNA fragment may, for example, become toxic to the cell when present in high levels. In this case, the best way to avoid any problems may be to use a low-copy vector.

A crucial step during the cloning process is the ligation of the DNA fragment to the plasmid. This is greatly facilitated by the use of specific restriction endonucleases. This means it’s important to check whether plasmid and selected restriction enzymes are compatible. In reality, this is not so problematic nowadays, as most modern vectors include an artificial stretch of DNA with many different restriction endonuclease cutting sites. If all else fails, blunt-end ligations are possible, but can be very difficult to complete successfully.


Features of the different Agrobacterium vectors

Two key advances made Agrobacterium transformation the method of choice for plant transformation.

  • Removal of all the T-DNA genes does not impede the Agrobacterium ability to transfer DNA but does prevent the formation of tumors. This allowed scientists to produce "disarmed strains."
  • The two main components for an Agrobacterium plasmid, the T-DNA and the virulence (vir) region can reside on separate plasmids. These components form the basis of modern Ti plasmid vectors, termed binary Ti vectors.

The T-DNA is approximately 10 to 30 kbp in size (about 10% of the plasmid size). Some plasmids can contain more than one T-DNA region. Furthermore, the plasmid contains about 35 virulence genes responsible for processing the T-DNA and its subsequent export from the bacterium to the plant cell.

Most in vitro gene manipulation techniques use E. coli to increase the plasmid yield, as it is a fast-growing bacterium. Consequently, one advantage of working with binary Ti vectors is their capability to replicate in both E. coli and Agrobacterium.

Let's talk about the different existing Agrobacterium vectors used in plant transformation.

Binary vector

Although the term binary vector literally refers to the entire system that consists of two replicons (DNA molecules), one for the T-DNA and the other for the virulence genes, the plasmid that carries the T-DNA is frequently called a binary vector. For convenience, I follow the literal definition of two plasmids to give more details to the reader.

A binary vector consists of two plasmids: a disarmed Ti plasmid carrying the T-DNA region and a helper plasmid containing the virulence genes.

The disarmed Ti plasmid:

The disarmed Ti-plasmid consists of a T-DNA and the vector backbone (Figure 2A). Both components have different genes that together determine the DNA sequence to be transferred and help in the identification of the transformed tissue or plant. Below I described each of these two components present in the disarmed Ti-plasmid.

Table 1. Components of the disarmed Ti-plasmid.

the right border (RB) and the left border (LB) can come from Octapine and Nopaline Ti plasmids.

Plasmid replication functions

It is an origin replication to be replicated in E. coli and A. tumefaciens. For A. tumefaciens we have IncP, IncW, pVS1 and pRi. For E. coli, we have pUC, ColE1, IncP, F factor, and Phage P1.

pUC and pBluescript plasmids contain multiple cloning sites.

Bacterial selectable marker

It can be kanamycin, ampicillin, gentamicin, spectinomycin, chloramphenicol, and tetracycline.

It can be kanamycin, hygromycin, phosphinothricin, glyphosate, phosphomannose isomerase

Plasmid mobilization functions

They support T-DNA mobilization functions such as OriT and Bom

It can be a GUS, LUC, GFP fluorescent proteins

Constitutive promoters usually drive the selectable marker genes. They include CamV 35S, T-DNA genes, ubiquitin, and actin genes.

The helper plasmid:

There are several virulence genes, named virA, virB, virC, etc., all producing different proteins that have different roles in the transfer of T-DNA from Agrobacterium to its host (Figure 2B). Below a chart describing the main functions performed for each gene.

Table 2. Description of the virulence genes functions in the Agrobacterium helper plasmid.

Sense phenolic compounds and induces expression of virulence genes

Important in the secretion system. Required for export of T-complex into the cell

Promotes high-efficiency T-strand synthesis

Topoisomerase and endonuclease functions, respectively

Chaperone function. Protect T-strand from nuclease attack.

Figure 2. Components of the binary vector. A) Disarmed Ti-plasmid. B) Helper plasmid. B marker: bacterial selectable marker LB: Left border Mob: Mobilization function MSC: Multiple cloning sites Ori A: replication function for Agrobacterium Ori E: replication function for E. coli P marker: Plant selectable marker Pro: Promoter RB: Right border Rep: Reporter gene.

Superbinary vector

The finding that some of the virulence genes exhibited gene dosage effects led to the development of a superbinary vector, which carried additional virulence genes. The superbinary vector system has the same backbone as the binary vector, but it also has an additional DNA segment that contains virulence genes like virB, virC, and virG from pTiBo542, and it is introduced into the small T-DNA-carrying plasmid. This region is also called "S vir". The additional vir genes have led to high efficiency in transforming various plants, especially recalcitrant plants, such as important cereals.

Figure 3. Components of the superbinary vector. S vir (in red) is the additional segment carrying virulence genes and it this differentiates with the binary vector.

Ternary vector

The ternary vector is a new system proposed by Anand et al. (2018) using a third plasmid— termed the “accessory plasmid” or the “virulence helper plasmid” —to carry an additional virulence gene cluster. The ternary vector system is a three-component system with a disarmed Ti plasmid, a helper plasmid, and an accessory virulence plasmid.

The disarmed Ti plasmid:

The disarmed Ti plasmid is composed of the T-DNA region flanked by the RB and LB, an origin of replication (ori) for Agrobacterium (e.g., pVS1 or pRiA4) and another ori for E. coli (pUC) and a plant selectable marker (usually kanamycin).

The helper plasmid:

The helper plasmid has a vir region, a bacterial selectable marker (str, streptomycin), tra/trb regions that function in conjugational transfer of the Ti plasmids, and, if present, a repABC/repAʹBʹCʹ region with function in replication of the Ti plasmids.

The accessory virulence plasmid:

The accessory virulence plasmid consists of an ori for Agrobacterium (e.g., pVS1 or pRiA4) and an ori for E. coli (pUC ori) and a bacterial selectable marker (spe, spectinomycin), and a large virulence region.

The ternary vector system nearly doubles the transformation efficiency in recalcitrant maize inbred lines (Anand et al., 2018). The ternary system also facilitated efficient Agrobacterium transformation of sorghum (Sorghum bicolor) and was used to develop transformation protocols for popular but recalcitrant African varieties (Che et al., 2018).

Figure 4. Components of the ternary vector. The accessory plasmid is the new component in the ternary vector and this differentiates with previous vectors. Tra/trb: transcriptional activators rep ABC: ABC replication origin.

Table 3. Commonalities and differences between the Agrobacterium vectors.

It has a helper plasmid containing virulence genes

It has additional virulence genes in the T-DNA-carrying plasmid

It works well in monocots and recalcitrant plants

It has an accessory helper plasmid containing a large virulence region

Causes an intense infection in some monocots and recalcitrant plants


What is a Plasmid Map

A plasmid map is a graphical representation of a plasmid, which shows the locations of major landmarks or elements of the plasmid. The relative positions of elements within a plasmid can be identified by restriction mapping. A restriction map is a map of restriction recognition sites within a particular plasmid. Hence, it is involved in the digestion of the plasmid by restriction enzymes. A restriction map is shown in figure 1.

Figure 1: Restriction Map

Two methods can be used in restriction mapping:

1. Restriction Digestion

– Restriction digestion of the plasmid with one or two restriction enzymes

– Mapping the plasmid based on the sizes of the fragments resulted from the plasmid

2. Sequencing

– Sequencing the whole plasmid

– Identification of the elements of the plasmid


RBSE Solutions for Class 12 Biology Chapter 15 Genetic Engineering

RBSE Class 12 Biology Chapter 15 Multiple Choice Questions

Question 1.
Which of the following enzyme cut DNA at a specific site?
(a) Ligase
(b) Polymerase
(c) Restriction Endonuclease
(d) All of the above
Answer:
(c) Restriction Endonuclease

Question 2.
Restriction endonuclease enzyme is found naturally in?
(a) Bacteria
(b) Virus
(c) Plants
(d) Animals
Answer:
(a) Bacteria

Question 3.
DNA Vector is?
(a) Plasmid
(b) cDNA
(c) Synthesized DNA
(d) All of the above
Answer:
(a) Plasmid

Question 4.
M13 is an example of?
(a) Plasmid
(b) Bacteriophage
(c) Cosmid
(d) All of the above
Answer:
(b) Bacteriophage

Question 5.
Which of the Blotting technique is used in the identification of DNA segments?
(a) Genomic DNA
(b) Western
(c) Southern
(d) Northern
Answer:
(c) Southern

Question 6.
Which enzyme joins free DNA ends?
(a) Restriction endonuclease
(b) Ligases
(c) Lysozyme
(d) All of the above
Answer:
(b) Ligases

Question 7.
Jumping genes are called?
(a) Phasmid
(b) Plasmid
(c) Cosmid
(d) Transposons
Answer:
(d) Transposons

Question 8.
Mullis discovered in 1989?
(a) Plasmid
(b) Polymerase chain reaction
(c) Southern Blotting technique
(d) Western Blotting technique
Answer:
(b) Polymerase chain reaction

Question 9.
C-DNA is used in the formation of?
(a) tRNA
(b) mRNA
(c) rRNA
(d) DNA
Answer:
(b) mRNA

Question 10.
Which of the following is a source of EcoRI?
(a) Bacteria
(b) Algae
(c) Plant
(d) All of the above
Answer:
(a) Bacteria

RBSE Class 12 Biology Chapter 15 Very Short Answer

Question 1.
Who is credited for developing recombinant DNA Technique (RDT)?
Answer:
Stanley Cohen, Herbert Boyer et al (1973).

Question 2.
Define recombinant DNA technology?
Answer:
Various effective measures required to incorporate changes in the DNA make up of any organism is called recombinant DNA technology.

Question 3.
What are cloning vectors?
Answer:
In recombinant DNA technology, in order to introduce the desired gene in the targeted plant/animal, a carrier is required which can carry the desired gene and can enter the targeted plant/animal and replicate it’s DNA. This carrier is called a vector. Plasmids, Bacteriophages and Cosmids are used as a vector in recombinant DNA technology.

Question 4.
What is meant by molecular probes?
Answer:
Segments of DNA or RNA with the help of which C-DNA or RNA segments of some organism can be identified are called molecular probes.
The molecular probes are of following types:

Question 5.
What are marker genes? Write examples.
Answer:
When the desired gene is incorporated with the vector, several unwanted products are also obtained. In order to eliminate these unwanted products and to identify the recombinant DNA in the host cell, a special type of gene is used. This produces special features in the modified/transformed cells. This gene which is incorporated in the vector DNA is called a marker gene.
Example: Kanamycin resistant gene.

Question 6.
What are reporter genes? Give examples.
Answer:
Besides marker genes, there are certain genes which produce or present some specific features in the host cell. These are called reporter genes. These genes produce a special effect on account of which the cells containing these genes look different from other cells.
Example: LUC gene found in fire-fly (“Jugnoo”) and produces bioluminescence.

Question 7.
What is the genomic library?
Answer:
Collection of the cloned segments of the entire genome of any organism is called the genomic library. A genomic library is formed by taking out complete DNA content of the haploid set of chromosomes of an organism.

Question 8.
What are Cosmids?
Answer:
Cosmids are a hybrid of plasmid and 2 (Lambda) phage. Such plasmids, in which DNA sequence of ‘Cos’ site of λ (Lambda) phage are inserted, are known as cosmids.

Question 9.
Define the restriction endonuclease enzyme.
Answer:
The enzyme which cut the DNA molecule at specific sites is called restriction endonuclease. These enzymes are just like molecular scissors, which cut DNA into the segment at specific sites.

Question 10.
Name the gels used in gel electrophoresis technique.
Answer:

Question 11.
Write a full form of RFLP.
Answer:
Restriction Fragment Length Polymorphism is abbreviated as RFLP.

RBSE Class 12 Biology Chapter 15 Short Answer Questions

Question 1.
What are cloning vectors? Write a brief account of various cloning vectors used in recombinant rDNA technology?
Answer:
After isolation of the desired gene(s), a vector is required which can incorporate this gene and along with it enter in the host cell & replicate it’s DNA in it. This vector is called cloning vector. Plasmid, Bacteriophages and Cosmids are main cloning vectors used in recombinant DNA technology.
Plasmids:

  • These are extrachromosomal components in the bacterial cell.
  • The DNA is a circular and double-stranded molecule.
  • They contain an origin of replication site and can replicate independently of a bacterial chromosome.
  • They have specific restriction sites where the desired gene can be incorporated.
  • They have marker sites.
  • The plasmid may contain 3 to one thousand genes.
  • The viruses which infect bacterially and cause lysis of bacterial cell are called bacteriophages.
    Example: λ (Lambda) phage and M13 phage etc.
  • Bacteriophages are a better vector as compared to plasmids.
  • Large DNA segments (24 Kbp) can be cloned in the bacteriophages.
  • Each bacteriophage produces a plaque in the culture. Hence their identification is easy.
  • This is a hybrid of plasmid and λ (Lambda) phage.
  • These can replicate in the host cell just like a plasmid.
  • Due to the presence of ‘Cos’ site, these are packed like phage particles.
  • Cosmids can be used to clone the DNA segment of up to 45 Kbp.

Question 2.
Comment on PBR 322 plasmid.
Answer:
PBR 322 plasmid is the most commonly used plasmid vector. In this plasmid, two marker sites TetR (Tetracycline resistant) and AmpR (Ampicillin resistant) are found. It contains recognition sites for 12 different restriction enzymes. Desired DNA (foreign DNA) is inserted in between TetR and AmpR gene with the help of restriction enzyme.

Question 3.
Write a short note on:

  1. Southern Blotting technique.
  2. DNA Fingerprinting.
  3. Polymerase Chain Reaction.
  4. Nomenclature of Restriction Enzymes.
  5. Features of Vectors.

Answer:
1. Southern Blotting technique:
This technique is used for the analysis of DNA segments. This was developed by E.M. Southern (1975) and hence named so. In this technique, the DNA segments are transferred on a nitrocellulose filter. These are then identified by hybridization with the DNA probes.

2. DNA Fingerprinting:
DNA Finger Printing was discovered by Alec Jeffreys and co-workers (1985). In this method DNA of a specific person is cut into small segments and is separated in the form of bands by electrophoresis. The identity of a person can be established by the specific base sequence found in the DNA of the person. This technique is used in resolving disputed paternity of any child and in detecting genetic diseases prior to the birth of a child. It is also used in the identification of criminals (Murderers and Rapists etc.)

3. Polymerase Chain Reaction (PCR):
Polymerase Chain Reaction was discovered by Mullis (1989). It is a powerful technique by which millions of copies of one DNA molecule can be obtained in a very short time without involving vectors for replication of DNA. This is performed in DNA thermal cycler. By repeating this cycle 20 – 30 times lacs of copies of DNA are obtained.

4. Nomenclature of Restriction Enzymes:

  • The first alphabet of the name of enzyme represents the name of the genus from which it has been isolated. It is written in capital letters.
  • The two alphabets after this represent the species of the genus. These are written in small letters.
    Note: All the three letters are written in italics.
    Example: E co = Escherichia coli.
  • The fourth alphabet (word) represents the strain of the species from which the enzyme has been isolated.
    Example Eco R = R strain of E.coli.
  • If more than one restriction enzymes are derived from the same organism (same strain), these are indicated by Roman numbers.
    Example: Eco RI, First restriction enzyme from R strain of E.coli. Eco RII, Second restriction enzyme from R strain of E.coli.
  • It can be inserted easily into the host cell and can be isolated easily.
  • It should freely self replicate in the host cell.
  • The vector must posses Restriction sites so that restriction enzyme can cut the DNA at that site so that the desired DNA fragment can be incorporated easily.
  • It should have one marker gene or marker site which can help in the selection of recombinants cells.
  • Transformation should be easy and perfect.
  • For expression of desired foreign DNA, the vector should essentially possess components such as a promoter, operator regulating sites.

Question 4.
Explain the process of forming the Genomic Library.
Answer:
Collection of the complete DNA content of a haploid set of chromosomes (genome) of an organism is called its a genomic library. A genomic library is formed by isolating entire DNA from a cell. The genomic library is formed by the following method:

Question 5.
Write a short note on the importance of Bacteriophage as a clonal vector.
Answer:
Bacteriophages are a better vector than the plasmids due to the following reasons:

  • It can clone DNA segment of relatively large size (24 kbp).
  • Every bacteriophage produces one plaque area in the culture through which testing is comparatively easy.

λ (Lambda) phage is more widely used as a vector than M13 phage because λ phage is having linear and double-stranded DNA and it is phage of E.coli and large-sized foreign DNA can be easily inserted in it’s DNA.

RBSE Class 12 Biology Chapter 15 Essay Type Questions

Question 1.
Write about different stages of techniques of recombinant DNA technology.
Answer:
1. Identification and Isolation of Desired Gene:
The desired gene or DNA segment is identified and for its isolation restriction endonuclease enzyme is used. Restriction endonuclease enzyme was discovered by Werner Arber and Hamilton O. Smith (1970).
Restriction Endonuclease:

  1. These enzymes are just like molecular scissors, which cut DNA at specific sites.
  2. These enzymes are found naturally in bacteria such as E.coli, Bacillus, Streptococcus and Thermus aquatics.
  3. Restriction endonuclease enzymes are of three types:
    1. Type I Endonuclease (RI)
    2. Type II Endonuclease (R-II)
    3. Type III Endonuclease (RIII). Type II.

    Note: Restriction endonuclease (RII) is most commonly used in gene cloning and restriction mapping.
    Example: E.co.RI, Hind II etc.

    Nomenclature of Restriction Endonuclease:

    1. The first alphabet of the name of the enzyme represents the genus from which it has been isolated. This is always written in capital letter.
    2. The two alphabets after this represent the species of that genus. These are written in small letters.
      Note: All the three letters are written in italics
      Example: Eco from Escherichia coli.
      Hin – From Haemophilus influenza.
    3. The fourth word represents the strain of the genus from which the enzyme has been isolated.
      Example: Eco R – From R strain of E.coli.
    4. In case more than one restriction enzymes are obtained from one organism, their number is indicated by Roman numbers.
      Example: Eco RI, (First from R strain of E.coli) Eco RII etc. (Second from R. strain of E.coli) Endonuclease enzyme Eco RI recognizes specific base sequence in DNA molecule and cuts between the base G and A.

    Note: When DNA segments obtained from two different sources are mixed together in the presence of DNA ligase enzyme, then both DNA segments are joined together by phospho-di-ester bonds and form a double-stranded structure.

    Other Enzymes used in Genetic Engineering:

    1. RNA dependent DNA Polymerase – This enzyme brings about polymerisation of nucleotides of DNA strand on an RNA template.
    2. DNA dependent DNA Polymerase – This enzyme brings about polymerisation of nucleotides of complementary DNA strand on a DNA template.
    3. Ligase – This enzyme joins the ends of DNA segments on the template.
    4. Lysozymes – These enzymes dissolve the cell wall of bacteria so that the bacterial DNA can be easily isolated.
    5. Alkaline Phosphatases – This enzyme cuts the phosphate at the 5′ end of circular DNA and helps to keep it in a linear form so that the foreign DNA segment can be inserted in it. This enzyme prevents the circular nature of DNA.

    2. Selection of Cloning Vectors:
    The desired gene is isolated with the restriction endonuclease. This desired gene containing a segment of DNA is then incorporated in a suitable vector. This vector should be able to enter into the target host or receptor cell and should replicate it’s DNA in the host cell.

    Essential features of Vector:

    1. It can be inserted easily into the host cell and can be isolated easily.
    2. It should freely replicate in the host cell.
    3. The vector must possess specific Restriction sites so that restriction enzyme can cut the DNA at those sites and the desired DNA fragment can be incorporated easily.
    4. It should have one marker gene or marker site which can help in the selection of recombinants cells.
    5. Transformation by it should be easy and perfect.
    6. For expression of desired foreign DNA, the vector should essentially possess components such as a promoter, operator regulating sites.

    Note: In E.coli both natural, as well as man-made vectors, can be used.

    Some important vectors used in E.coli are:

    1. Plasmid:
    Lederber, (1952) first observed the plasmids as extrachromosomal material in Bacterial cell. It has the following important features.

      1. These are extrachromosomal (Other than chromosomal DNA) components.
      2. These are circular, double-stranded DNA molecules
      3. They contain an origin of replication (ori). Hence being able to replicate independently within the cell.
        1. These are not necessary for the growth and survival of Bacteria
          .
        1. These contain specific restriction sites where the desired gene can be inserted.
        2. Marker genes or marker sites are also found.
        3. The plasmid may contain three to one thousand genes.

        Note: The Most commonly used plasmid vector is pBR322.
        In this plasmid, two marker sites TetR (Tetracycline resistant) and AmpR (Ampicillin resistant) are found. It contains recognition sites for 12 different restriction enzymes. Desired DNA (foreign DNA) is inserted in between TetR and AmpR gene with the help of restriction enzyme.

        To insert the foreign DNA in the plasmid it’s DNA is made linear by use of restriction enzyme and after that in between the two ends of the DNA of plasmid, foreign DNA of 5-10 Kb is inserted or incorporated.

        Note: Depending upon the condition of replication the extra chromosomal material found in the bacterial cell is called plasmid or episome. The extrachromosomal structure capable of replicating independently is called plasmid but if it replicates only when joined with the bacterial chromosome along with it, then is called as an episome.

        2. Bacteriophage:
        The Viruses which infect Bacteria are called Bacteriophages.
        Example: λ (Lambda) and M13 etc.
        Bacteriophages are a better vector than plasmids due to the following reasons:

        • The DNA fragment of large size (24Kb) can be cloned in these.
        • Every bacteriophage produces one plaque area through which testing is comparatively easy.

        λ phage is more widely used as a vector than M13 because of the following:

        • They are bacteriophage of E.coli.
        • DNA is linear and double-stranded.
        • Non-essential part of a phage DNA can be removed. As a result, the size of the vector molecule is reduced hence large-sized foreign DNA can be easily inserted in it.

        • It is a hybrid of plasmid and lambda (λ) phage.
        • It was first of all constructed by Barbara Horn and John Collins (1978).
        • Such plasmids, in which DNA sequences of ‘Cos’ site of lambda phage DNA are inserted, are known as cosmids. Hence the cosmid possesses the features of both plasmid as well as the lambda bacteriophage.
        • Cosmids replicate in the bacterial cell just like the plasmid and on account of the presence of ‘Cos’ site, these are packed like lambda phage particles.
        • Cosmids can be used to clone DNA segments of up to 45 Kbp in size.
          Example: Cosmid PLFR-5
        • There are present two Cos sites, 6 restriction endonuclease sites, the origin of replication site and tetracycline antibiotic resistance genes, in this cosmid.

        Note: Besides plasmid, bacteriophages and cosmid, there are certain other vectors also being used in genetic engineering.

        Transposons are also known as Jumping genes.

        3. Insertion of the desired gene in the cloning vector:
        For insertion of the desired gene into a cloning vector, first similar restriction sties are made in both the genes. After this, both are joined together (as both have the same restriction sites). This process is called Ligation.
        In this process following enzymes are required:

        1. Restriction endonucleases
        2. Methylases
        3. DNA ligases
        4. Alkaline phosphatases
        5. Reverse transcriptase
        6. Terminal transferases

        4. Multiplication of recombinant DNA in host cell:
        Recombinant DNA may be inserted into the host cell by following two methods:

        In this process, E.coli bacterial cell is mainly used as the host. These processes (Transformation and Transduction) have been studied earlier. The Recombinant DNA multiplies in the E.coli cells

        5. Identification of the cloned gene and its transfer in other organisms:

        • We are using certain genes such as antibiotic-resistant gene or LacZ gene (which produces β galactosidase) as marker genes so that if cloning sites are present in these genes and DNA is inserted in between them the genes become non-functional and called as insertional inactivation.
        • On the basis of expression or non-expression of a particular gene as a marker gene, we can select recombinants from non-recombinants.
        • Besides marker genes, there are certain other genes which produce specific features. These are called reporter genes which look entirely different from other cells due to special features.
          Example: LUC (Luciferase) gene which is found in firefly and produces Bioluminescence.

        Question 2.
        Describe different blotting techniques in details.
        Answer:
        Blotting Technique:
        In this technique, DNA segments are derived on agarose gel by electrophoresis and are then transferred and stabilised on a nitrocellulose filter. These are then identified by hybridization with DNA probes. This process is called blotting technique.
        1. Southern Blotting Technique: E.M. Southern (1975) first used this method and transferred DNA segments on the nitrocellulose filter. This technique was named a Southern blotting technique. By this technical analysis of DNA, segments are done.

        2. Northern Blotting Technique:
        Alwin et al (1979) transferred RNA segments after electrophoresis on amino benzyl oximetry (OBM) membrane in place of nitrocellulose filter. This technique was named as Northern blotting technique. This technique is used for the analysis of RNA segments.

        3. Western Blotting Technique:
        Tobin et al. (1979) first break down proteins into polypeptides with the help of sodium-do-decyl sulphate (SDS). After separating them with the help of electrophoresis, transferred these on nitrocellulose filter or nylon membrane. These were then decoded on an X-ray plate and identified the protein. By this technical analysis of proteins is done. This is called western blotting technique.

        Question 3.
        Write a detailed account on plasmid as a cloning vector.
        Answer:
        Plasmid:
        Lederber, (1952) first observed the plasmids as extrachromosomal material in Bacterial cell. It has the following important features.

        • These are extrachromosomal. (Other than chromosomal DNA) components.
        • These are circular, double-stranded DNA molecules.
        • They contain an origin of replication (ori). Hence being able to replicate independently within the cell.
        • These are not necessary for the growth and survival of Bacteria.
        • These contain specific restriction sites where the desired gene can be inserted.
        • Marker genes or marker sites are also found.
        • The plasmid may contain three to one thousand genes.

        Most commonly used plasmid vector is pBR322.
        In this plasmid, two marker sites TetR (Tetracycline resistant) and AmpR (Ampicillin resistant) are found. It contains recognition sites for 12 different restriction enzymes. Desired DNA (foreign DNA) is inserted in between TetR and AmpR gene with the help of restriction enzyme.

        Question 4.
        What do you understand by molecular probes? Explain its utility.
        Answer:
        Molecular Probes:
        Segments of DNA or RNA with the help of which cDNA or RNA segments of any organism present in it can be identified are called molecular probes.
        The molecular probes are of two types.

        • These are used to identify specific DNA segments used in the research of genetic engineering.
        • Pollutants in food can be detected with the help of probes.
        • These are used in the field of forensic science, in resolving disputed paternity issues and establishing the family relationship.
        • These can be used to identify an improved variety of crops and hybridized seeds of crops.

        Question 5.
        Write an essay on the importance of genetic engineering.
        Answer:
        Scientist working in the field of biotechnology have succeeded in achieving results which have proved advantageous in the field of medical science, agriculture and industries. By using these techniques it has become possible to improve the variety of agricultural crops and domestic animals and also the quality of industrial products. Presently biotechnology is considered as the most important branch of biology.
        Some important achievements are as follows:


        Plasmid vs Lentivirus as a transfection vector - Opinions? (Jan/12/2008 )

        I'm looking into creating a stably transfected variant of a few human tumor cell line that I'll eventually use for xenograft experiments. I'd like to insert a tag for in vivo imaging (IVIS). I'm thinking GFP or luciferase would be suitable, but I'm not sure what is a better vector. I have a pEGFP plasmid left over from a earlier student in the lab that I could use with lipofectamine, but someone suggested a lentiviral GFP would be better. My specific questions are:

        Would I expect a difference in transfection stability, assuming G418 selection?
        After selecting G418-resistant clones, can I remove the G418 before xenograft?
        Would one or the other have a greater trasfection efficiency?

        Any opinion would be appreciated! Cheers-JAH

        lentivirus are good for cells that are hard to trasnfect using lipids. the choice of reporter system depends on which imagine system do you have. Luc is better and more quatitative.

        For your specific questions:
        1) stability is more or less gene specific. ie. is the gene you want to express grwoth promoting or inhibitory. Cells will drop the inhibitory gene over time if you dont use a regulated expression system. Both routes can generate stable cell lines.

        2) Yes, you can remove selection pressure for a few generations.

        3) Its cell type dependent. Lenti have more chance to work but making virus is more exprensive and takes longer to get the virus.

        Would I expect a difference in transfection stability, assuming G418 selection?
        i don't strickly remember the mechanism of virus insertion in genomic dna but i still belive that the DNA is circular or should be opened for linear insertion. In viruses, the opening point is quite strict, and does not interferes either with your selection gene or the gene of interest. So it's better than plasmids in this way, as the opening point wich is not compatible with the selectio marker may occur in the gene of interest.

        After selecting G418-resistant clones, can I remove the G418 before xenograft?
        yes you can remove it but for few generations as mentioned previously.

        Would one or the other have a greater trasfection efficiency?
        it depends on the cell type. for hela 293 and such cells, transfection by plasmids gets a high efficiency. On primary cell lines, transfection has a poor efficiency and lentiviruses have better delivery efficiency in general. so retroviruses are better choice (if you can afford costs)

        My preference would be to go with plasmid transfection than lentiviral infection as virus preps are more time consuming and an additional step. Also to choose between GFP and luciferase, it depends on the experiments you wish to do. if you wish to use them for invivo, then having gfp will help in easy readout. But something quantititaive, then luc might be better.

        In terms of stability, both methods should be kind of similar.

        One can get high transfection efficiency with both systems, ofcourse depending on cell types. but something like 293 or hela, it should be fine.

        Thanks again for all of your comments. I think for my purposes, I don't necessarily need quantitative data, so I think GFP will be sufficient. Basically, I'm orthotopically inoculating human rhabdomyosarcoma cells into the thigh muscle of nude mice and would like to be able to verify tumor engraftment after injection and localize metastases, should they occur I'd also like to perform FFPE histology with the affected tissues, so I think GFP will facilitate this better than a luciferase tag. I've not tried to transfect anything into these cells so I don't know yet if thay are as genehunter-1 put it "hard to trasnfect using lipids". Reading these responses, I'm inclined to try the plasmid vector first As such a few more questions came to mind.

        "Yes, you can remove selection pressure for a few generations." (genehunter-1, regarding G418 selection)
        "yes you can remove it but for few generations as mentioned previously." (fred_33, regarding G-418 selection)

        1) I can't very well put G418 in to mice though. should I just wean the selected cells off of the G418 prior to inoculation, and just hope they don't kick out the plasmid?

        Quoth fred_33:"i still belive that the DNA is circular or should be opened for linear insertion. So it's better than plasmids in this way, as the opening point which is not compatible with the selection marker may occur in the gene of interest."
        1) So, should I compare a linearized plasmid in parallel with a circular/supercoiled one?
        2) Assuming the plasmid is linearized by RE digestion, is there a particular end (blunt/sticky/overhang) that I should strive for?
        3) Where should the digestion site be. obviously not through the GFP or selection gene or their promoter/regulatory regions so as not to alter the ORF?

        Quoth genehunter-1 "is the gene you want to express grwoth promoting or inhibitory. Cells will drop the inhibitory gene over time if you dont use a regulated expression system. "
        - I don't believe the plasmid I have is explicitly responsive to growth regulatory process. I believe the promoter was cloned from a 'housekeeping' gene, though I'm trying to figure out what this regulatory element is. Anyway, the cells I'm going to try are show very aggressive growth, doubling in

        18h, and do not appear to be contact inhibited at all. But I'm not sure if the plasmid harbors something that might change this behavior, but will be sure to compare them vs. non-transfected cells before in-vivo use.


        A plasmid is an extrachromosomal DNA of bacteria, yeasts, archaea and protozoa. They are small double-stranded circular DNA molecules. Whereas, a vector is a small DNA molecule that acts as a vehicle to deliver foreign DNA from donor to host. So, this is the key difference between plasmid and vector.

        Moreover, a further difference between plasmid and vector is that the plasmids are naturally occurring in bacteria and other organisms, but some vectors are natural while some are artificially synthesized.

        Below infographic summarizes the difference between plasmid and vector.


        The Next Plasmid

        Interestingly, the biologists who do not think they need design tools are often craving better tools for supporting their laboratory efforts. Tools for primer design, for planning of cloning experiments, and for sample tracking are very high on their wish list. This may partly explain their lack of interest in the design aspect of their work.

        The vast majority of potential users of design tools have been trained to clone DNA. They are completely focused on the process of deriving new DNA molecules from existing ones.

        When they say that their project is simple, they don't mean that it will be simple to deliver an expression system that works. What they mean is that it is simple to figure out the next plasmid to make.

        For instance, if it is easy to add a solubility tag to the coding sequence of a gene in an existing plasmid, then they assume there is nothing more to think about. The next plasmid to make is easily identified mostly based on the ease of cloning criteria, not on functional criteria.


        Discussion Questions

        1. Why was it important to find an enzyme that would cut the plasmid at only one site? What could happen if the plasmid were cut at more than one site?

        2. Which restriction enzyme did you use? _____________ Ask other groups what they used and compare the final transgenic plasmids. Why might there be some of different lengths?


        3. Why was it important to discard any enzymes that cut the plasmid at the replication site?

        4. Why is it important to cut the plasmid and the human DNA with the same restriction enzyme?

        5. Do restriction enzymes exist naturally in organisms? If so, what is their purpose?

        6. Why would restriction enzymes that created "blunt" ends not be as useful in recombination as those that create sticky ends?


        7. In the activity, you simulated creating a recombinant bacteria organism. For each of the following materials, indicate what they represent?

        Scissors _______________________________________Tape ______________________________________



        Watch the video: Plasmids. Genetics. Biology (July 2022).


Comments:

  1. Draven

    Thank you for the information. I did not know this.

  2. Gaderian

    sorry, I deleted this message

  3. Ordland

    Is she serious?

  4. Brangore

    In my opinion someone here is obsessed

  5. Chayson

    On mine it is very interesting theme. I suggest you it to discuss here or in PM.

  6. Kazrami

    It seems to me an excellent idea

  7. Calbhach

    I saw something similar in English-language blogs, on the Runet about this somehow you will not see posts very often.



Write a message