qbm chapter 15

viral vector delivery

A gene of interest is inserted into the viral genome and eukaryotic cells are infected with the vector In the case of retroviruses, once inside the cell, the viral RNA is converted into DNA by the viral reverse transcriptase and both the virus and the gene of interest are integrated into the host genome Adeno-associated virus can also integrate its DNA into a specific location on chromosome 19

Bacteriophage Lambda

A type of vector that played a very important role in early molecular biology and genetics research Has a 45,000 bp genome, and a central 15,000 bp can be removed by restriction enzymes and replaced with 10-20,000 bp of DNA

gene of interest

A very good initial screen for clones carrying the correct vector, and it also prevents most contaminants from growing on the plate, but other techniques must be used to find the clones that contain the

national center for biotechnology information

An electronic library of the human genome, obtained from both the human genome project and independent researchers submitting single genes at a time, is available at the

protein expression or gene mapping

BACs and YACs contain elements required for replication in bacteria (ori) or yeast (ARS, centromeres, telomere) and they can be used for

in vitro packaging

Bacteria can also be used to deliver DNA via transduction into E. coli, in a process called

T4 DNA polymerase

Blunt end digestion offers versatility, in that two different blunt digestions can be ligated together, and overhang digestions can be filled in or chewed back to blunt ends using ___________________ and ligated together

white

Clones with the vector that has the B-galactosidase gene disrupted by an insert (gene of interest) will remain

screening gene libraries to identify the clone that is carrying the gene of interest - clones such as bacteriophage are spread onto dozens of large agar plates and screened with a radiolabeled probe

DNA hybridization is useful for

CaCl2

E. coli cells must undergo chemical treatment with _________, which partially disrupts the cell membrane

antibiotics

Genes that confer resistance to various __________ are frequently used as selective markers in cloning vectors (ampicillin, tetracycline, neomycin, kanamycin)

cDNA library

If a researcher is interested in only working with the spliced gene, called the coding sequence, a _______________ can be generated contains the entire coding sequence without introns or upstream regulatory regions

antisense

If the two individuals express the same gene, the cDNA from one will hybridize with the mRNA from the other, since the two are ________ to one another

partitioning

In addition, some species require a _____________ sequence, such as the centromere in eukaryotes

re-ligate

In both single overhang and blunt digestions, the plasmid can ____________ to itself or multiple inserts can ligate to one another, forming larger plasmids than expected and reducing efficiency

SV40

In mammalian cells, virus-based origins such as ________ can be used for vector replication

origin of replication

In order to be replicated and maintained stably in an organism, each chromosome must contain at least one _____________________ , and it should not be removed by digestion

sample

In this technique, the mRNA from one individual is converted into cDNA, and the mRNA from the a second individual is added to the

ARS

In yeast, this sequence is called the ________, which consists of 11 base pairs that recruit replication proteins

Antibiotic Resistance

Many plasmids carry a gene for antibiotic resistance and any bacteria that take up the plasmid can grow in the media containing the antibiotic Therefore, only cells containing the vector of interest should grow on the plate, and those that do not will be killed by the antibiotic

DNA uptake

Membrane disruption by CaCl2 allows

entire genes in their natural settings (such as when studying introns, regulatory regions, and intergenic DNA) or to try to isolate a gene for the first time if only part of the sequence is known

Molecular biologists use genomic libraries to study

cDNA

Must insert ______ (not genomic DNA) in bacteria for protein expression systems

Restriction Mapping

One of the most common ways to determine if a cloning experiment was successful is to verify that both the vector and insert are present in a clone using a miniprep, restriction enzyme digestion, and agarose gel electrophoresis A colony is picked from a plate, grown overnight, and miniprepped to obtain purified plasmid DNA

blue/white screening

One way to identify whether a clone is carrying the gene of interest via phenotypic screening, is to use the lac operon in a process called

selectable marker

Only cells that contain plasmids with the __________ ___________ can survive

DNA probes

Phage containing the gene of interest can be screened using

replicons

Plasmids are considered ___________, or pieces of DNA that are able to function like a chromosome in vivo and are replicated, partitioned, and maintained over many generations

Genomic DNA → transcription, splicing to remove introns, and mRNA purification → reverse transcriptase creates a DNA-RNA hybrid → digest RNA, generate dsDNA → cDNA library

Recall the process of cDNA library generation.

vectors

Replicons or plasmids that are designed to "carry" DNA sequences of interest and facilitate their manipulation and study, are called

origin of replication and a selectable marker

The E. coli component of a yeast shuttle vector includes an

gene of interest

The bacteriophage inserts its DNA along with the inserted gene into the host cell, which replicates the

Phenotypic Screening

The most common method of identifying the clone Is used when a plasmid or cloned gene changes the phenotype of the cell in an obvious way (making the cell resistant to an antibiotic, or changing its color) Usually more than one technique is used, such as first screening with antibiotic resistance followed by verification with a restriction digest, colony PCR, or DNA sequencing

prevent the plasmid from reannealing (reducing false positives) increase ligation efficiency due to the complementary base pairing of the sticky ends and ensures directionality of the insert during ligation step

The multiple cloning site and gene of interest are usually digested with two different restriction enzymes that produce different overhangs to

white

The multiple cloning site is located within the lacZ gene. If a gene of interest is inserted into the multiple cloning site, it will disrupt the lacZ gene, preventing expression of B-galactosidase, and causing bacterial colonies to be

single large band on the gel

The plasmid is digested with restriction enzymes at sites on each side of the gene of interest and run on a gel: Clones that contain only the plasmid (with no inserted gene) will show a

Viral

The rDNA will be packaged in phage particles and mixed with E. coli and plated. The phage will infect the bacteria and make many copies of the gene. ________ plaques are areas of many cloned phage carrying the gene of interest

oriC, a region rich in AT repeats that is recognized by the replication initiator protein DnaA

The replication machinery is assembled at specific DNA sequences; in E. coli this is called the

autonomously replicating sequence (ARS), a yeast centromere (CEN), and a yeast selectable marker

The yeast component of a yeast shuttle vector includes an

first

This DNA-RNA hybrid is separated from cDNA that did not hybridize, and this remaining cDNA is now a representation of expressed genes specific to the ________ individual

vector

This does not guarantee that the cells are also carrying the gene of interest, only that they contain the _____ with the antibiotic resistance

two bands on a gel

Those that contain the plasmid and gene of interest will have

lambda (λ) phage

To accomplish this, a portion of the _______________________ genome is removed with a restriction enzyme and replaced with the gene of interest; the resulting recombinant gene is packaged, mixed with E. coli, and plated

an origin of replication multiple cloning site selectable marker

To be useful for DNA manipulation or protein expression, vectors must have:

examples of non-conditional mutants

URA3- yeast lack orotidine-5'-phosphate decarboxylase and are therefore unable to synthesize uracil, which must be provided in the media. But since these mutant yeast cells do not contain the enzyme, they also survive in the presence of 5-FOA E. coli B834 cells cannot synthesize methionine on their own and are non-conditional mutants because they cannot survive without methionine supplement

Gene Library Screening

Various strategies were developed (splitting the library up and testing sections, developing degenerate probes), but sequencing the entire human genome was more practical

human genes in eukaryotic systems, to create vaccines, and as delivery systems for gene therapy

Viral vectors are used to study

CaCl2 treatment and heat shocking of cells is cost-effective, but it takes several hours to perform and the results are not known until the following day. Electroporation is faster and provides immediate feedback as to whether the transformation was likely successful or not, but it is more expensive and the cells take longer to prepare.

What are the advantages and disadvantages of chemical treatment with CaCl2 and electroporation?

A cell that has been modified to take up DNA from the environment via transformation.

What is a competent cell?

blue

When both IPTG and X-gal are present in the media, the cells with the vector express B-galactosidase which cleaves X-gal, and the resulting insoluble product turns the cells

The hosts used for cloning have been modified to efficiently take up DNA, lack restriction enzymes, lack proteases, lack recombination, and sometimes contain other manipulations to promote cloning and protein expression, such as some strains carrying T7 RNA polymerase on their genome. These are features that are unlikely to exist in conjunction in nature.

Why must special host cells be used for cloning?

gene library

a collection of all the genes in an organism (such as in vectors), and it may or not may include intros of intergenic DNA

cosmids

a combination of a plasmid and lambda phage and contains an origin of replication and cos sites used for phage packaging can replicate as a phage, in E. coli, and in eukaryotes if they have the appropriate origin

PCR

a method to identify the clone where the clones are heated and the plasmid is amplified with specific primers to detect the presence or absence of the gene of interest only the linear insert band would be visible, as the plasmid is not amplified in this technique

cloning

a standard __________ vector, like pBluescript or pGEM-T are smaller designed for efficient uptake and replication in the host cell High copy number, many restriction sites, blue/white screening, multiple antibiotic resistances

heat shocked at 42 °C for a short time, then chilled on ice

after DNA uptake, the cells are then mixed with the plasmid and

An origin of replication (ori)

allows the plasmid to be replicated and maintained over multiple generations in E. coli

examples of conditional lethal mutants

bactericidal antibiotic selection, where bacteria that contain the plasmid will survive, and those that do not will die yeast cells that die in the presence of 5-FOA wild-type yeast have the URA3 gene that encodes orotidine-5'-phosphate decarboxylase, an enzyme that catalyzes orotidine monophosphate to uridine monophosphate, a nucleotide found in RNA wild-type yeast also convert 5-FOA into 4-fluorouracil (a toxic compound), and therefore die in the presence of 5-FOA, whereas mutants lacking the enzyme survive

Ca2+

binds to the negatively-charged DNA backbone, allowing it to cross the bacterial membrane and neutralize the DNA

purify and study proteins

cDNA libraries are especially useful for researchers who wish to

Viral Vectors such as retroviruses (like HIV or lentivirus) or adeno-associated virus (AAV)

can also be used as delivery systems for genes, and they are much more efficient than standard transfection (eukaryotic transformation) with chemicals

Plastids/Chloroplast DNA

can also be used by scientists who work with plants replicated separately from the plant genome This DNA is generally not spread by pollen, which is important to prevent the spread of genetically modified organisms

Antibody Screening

can also be used to screen colonies of cells, to determine whether they are expressing a protein from the vector This is not commonly used, since most vectors used for cloning are not designed to be expression vectors

DNA Hybridization

can be used if part of the gene of interest's sequence is known Cells on a plate can be transferred to a nylon membrane, lysed, treated to produce ssDNA, and washed with a radiolabeled probe which can hybridize to the gene of interest The developed X-ray film can be compared to the original agar plate, and the colony containing the gene of interest can be located

A cDNA subtraction library (or subtractive hybridization)

can be used to study differences between two organisms

shuttle vector

can replicate in multiple hosts (one that replicates in both E. coli and yeast would require an ori and an ARS)

the pores or invagination in the cell membrane cells are plated and grown overnight, so the success of the transformation is not immediately known

chemical treatment forces the plasmid into the cell through

expressing specific genes in plants (like genes for herbicide, disease, and drought resistance), or expressing proteins or medicinal compounds

chloroplast DNA are useful for

plasmids

circular dsDNA that replicate independently of the E. coli genome, and often carry important genes such as antibiotic resistances

Genomic library

contains all of the DNA of a cell, in short fragments an organism's entire genome can be digested into thousands of pieces, ligated into a vector, cloned, and the gene of interest can be located

multiple cloning site, or polylinker

contains the gene of interest contains several unique restriction enzyme digestion sites next to one another; this allows the gene of interest to be inserted into the plasmid at this location via various unique restriction sites present

gene libraries

cosmids can accommodate up to 52,000 bp of DNA and are useful for creating

In this case, the plasmid contains the gene for the lac repressor, which is synthesize and binds to the operator, preventing transcription of lacZ, a reporter Upon the addition of IPTG to the media, the lac repressor is removed, allowing transcription of lacZ This gene encodes B-galactosidase, a protein that breaks down galactosidase into monosaccharides; it can also metabolize other substrates, such X-gal

describe how the lac operon is used in blue/white screening

This technique requires special equipment and expensive cuvettes with electrodes, and the cells take longer to prepare, since salt (which can affect the process) must be completely removed; however, large preparations of cells can be made and stored at -80°C for future use

disadvantage of electroporation

Larger, so less efficient transformations, and fewer restriction sites, therefore genes are often cloned into cloning vectors first, and then digested and ligated into expression vectors

disadvantages of expression vectors

non-conditional mutants

display the mutant phenotype under all conditions, and require a supplement to survive

time constant

electroporation only takes seconds and provides a ________ _________ that tells the researcher whether the transformation was likely successful or not

selectable marker

ensures that the bacteria that grow on the plate or in the media are only those that have taken up and maintained the vector, and any non-transformants will not grow or be killed

some yeast are temperature-sensitive mutants and grow at 30°C, but not at 37°C

example of conditional mutants

Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)

have been developed to carry hundreds of thousands or millions of base pairs of DNA

E. coli cell lines

highly efficient, grow well, easy to regulate, take up foreign DNA, no proteases, restriction enzymes, or recombination, allow for many plasmids, high/low mutation rates

They are created by first purifying cellular mRNA, and then using reverse transcriptase and an oligo-dT primer that is specific for the mRNA polyA tail to generate a DNA-RNA hybrid The RNA strand is then digested with RNase H, and DNA polymerase can generate dsDNA from this ssDNA template This DNA, if inserted into an expression factor, can produce the exact protein encoded by the gene

how are cDNA library's created

To generate a genomic library, the genome is lightly digested with a restriction enzyme to generate fragments and is placed in a host cell The host now contains a vector, carrying a fragment of the original genome, which can be screened to locate the gene of interest using a labeled probe Plasmids, bacteriophage, cosmids, BACs, and YACs are all used to generate genomic libraries

how do you generate a genomic library?

"plaques"

in bacteriophage lambda, DNA of interest is digested into fragments of this size, ligated into the phage genome, packaged into phage, and mixed with E. coli This mixture is spread on agar plate, where the phages will infect the E. coli and make millions of copies of themselves and the inserted gene Small circular __________ will appear on the plate, which are zones of phage and lysed E. coli

expression vector

like pGEX or pET, which allows for high levels of protein expression in E. coli

Chemical Treatment

method to induce competency in cells cells can be treated with chemicals that partially disrupt the cell membrane, rendering it permeable to added DNA molecules

Natural Competence

method to induce competency in cells some strains of bacteria, such as H. influenzae and B. subtilis (but not E. coli) are naturally able to take up DNA under certain conditions natural competence of certain bacterial cells and the transformation of these cells with DNA are historically important because they demonstrated that DNA is the genetic material (Griffith, Avery)

electronic library or database - This library is usually accessible online to anyone with internet access

most genomic libraries are stored in an

DNA sequencing

must be performed on all vectors to definitively verify that the gene is actually present and lacking mutations

Conditional lethal mutants

mutants that can grow under permissive conditions but die under restrictive conditions

Conditional mutants

mutants that can grow under permissive environmental conditions, but cannot grow under different restrictive or non-permissive conditions

Cl-

partially disrupts the membrane of E. coli by causing it to swell and create pores

phagemids

plasmids have an f1 phage origin that allows for packaging in phage requires a pilus still limited to ~4000 bp of DNA, but can allow for more plasmid copies as its not under bacterial control

auxotrophs

some cells require the addition of a specific metabolite to the media to survive (called ________________), such as E. coli B834 cells which cannot synthesize their own methionine and can therefore be used for 35S-methionine and selenomethionine labeling of proteins

Bacterial vectors that propagate in E. coli

the most common system for basic cloning and expression experiments, but they are usually limited to taking up inserts under 10,000 base pairs, due to the transformation efficiency of large plasmids

E. coli DH5α, DH12S, JM109, E. cloni, TOP10, and XL10-GOLD

used for cloning because of modifications specific to each cell line, such as high transformation efficiency, high plasmid copy number, lack of restriction enzymes, lack of recombination, a disrupted lacZ gene, certain antibiotic and phage resistances, expression of the lacZΩ subunit for α-complementation, and other deviations from wild-type. These cells typically have a high transformation efficiency

other cell lines, such as E. coli BL21(DE3)

used for protein expression because they contain the T7 RNA polymerase gene in their genome and lack proteases that would digest recombinant proteins some expression lines contain the T7 lysozyme gene to reduce background expression, and some competent cells have been further improved for mRNA stability, for the expression of toxic genes, or for use with low copy number plasmids

Electroporation

used to shock E. coli or mammalian cells with an electric current, causing the membrane to be briefly disrupted and form pores to take up DNA Can also be used with eukaryotic cells

blunt end digestions

used when the necessary restriction sites aren't available on the vector or insert

E. coli BL21(DE3) E. coli B834 Tuner Rosetta (mammalian codons) Mach1

what are the expression strains