Cloning Genes for Synthetic Biology (Chapter 7)

Often, vectors have been designed to provide some convenient means to perform the following:

1. A mechanism to select host cells containing the vector 2. Ability to insert genes into the vector 3. Detect the presence of an inserted gene in the vector.

What are the antibiotics commonly used in synthetic biology (cloning)?

1. Ampicillin 2. Kanamycin 3. Chloramphenicol 4. Streptomycin 5. Tetracycline

What are the key features of cloning vectors?

1. Antibiotic resistance gene 2. Selectable marker 3. Promoter region 4. origin of replication (ORI) 5. Multiple cloning site (MCS) or polylinker 6. Insert 7. Primer binding site 8. Terminators

Name three methods of getting vectors into mammalian cells:

1. Calcium phosphate precipitation 2. Lipid-mediated transfection 3. Viral transduction

What are the properties of cloning vectors?

1. The vector should be reasonably small and manageable DNA molecule. 2. Moving the vector from one organism to another should be relatively easy. 3. generating and purifying large amounts of vector DNA should be straightforward.

A cloning vector is any molecule of _________ that can replicate ____________ inside a cell and is used for carrying cloned ___________ or segments of DNA. Usually a small multicopy _____________ or a modified virus.

DNA, itself, genes, plasmid

Key Features of Cloning Vectors: The origin of replication contains the ______ sequences that are essential for the bacterial to make copies of the ____________. there are many different ________, and slight DNA sequence differences result in a profound difference in how many ____________ of the plasmid are ___________ in each cell. The origin controls how many ___________ are present. In addition, the sequence of the origin of replication _____________ when the plasmids are ____________. ____________ plasmids replicate when the bacteria undergo cell division and are then _______________ into each daughter cell. ______________ plasmids can replicate any time in the bacteria growth cycle. The origin also determines whether or not two ______________ plasmids can be _____________ in the same bacterial cell. Plasmids with the same type of origin are _____________ and usually one plasmid is ______________ from the bacterial cell.

DNA, plasmid, origins, copies, maintained, copies, determines, replicated, stringent, partitioned, relaxed, different, maintained, incompatible, rejected

A vector in molecular biology is a molecule of _________ which can ____________ and is used to carry cloned __________ or DNA ______________.

DNA, replicate, genes, fragments

In recombineering, _________ is used to create ___________ fragments of DNA that have ends ___________ to regions of a bacterial artificial chromosome (BAC). Bacteria containing the genes for the _________ proteins are grown at _________ temperature for a few hours to ___________ production of the _______ proteins, which then _____________ the ends of the insert and the region on the vector that has the _______________ sequence. The enzymes ______ the vector DNA and attach the __________. Final bacteria are grown at lower temperatures to ____________ any further _______ gene expression.

PCR, linear, homologous, Red, high, induce, Red, recognize, homologous, cut, insert, inhibit, Red

Lambda is a ____________ that infects E. coli with its dsDNA _____________. It is capable of ____________. This is a state in which a virus ___________ into the bacterial ___________ (prophage). The prophage is _________ with the host genome without making virus ___________ or destroying the host cell. Excision of lambda ____________ from host genome is necessary to make virus particles (lytic phase).

bacteriophage, genome, lysogeny, integrates, genome, replicated, particles, genome

Alpha complementation is the assembly of functional _______-____________ from ___-terminal alpha fragment plus rest of protein. Alpha fragment is the ___-terminal of beta-galactosidase. Beta-galactosidase protein is unique since it can be expressed as two ___________ that come together to form a functional ____________. The alpha fragment can be expressed from a ___________ and the remainder can be expressed from the host ______________. If the plasmid and chromosomal gene segments are _________, they produce two protein ___________ that associate to give an active ___________.

beta-galactosidase, N, N, pieces, protein, plasmid, chromosome, active, fragments, enzyme

Reporter Genes: lacZ One common reporter gene is the lacZ gene which encodes ______-_______________. This enzyme normally splits ____________ into ______________ and ____________. However, this enzyme will also split a wide range of __________ compounds. The two most commonly used artificial galactosides are __________ and ___-_______. O-nitrophenyl galactoside (ONPG) is split by _____-_____________ into ___-____________ and _______________. The o-nitrophenol is __________ and soluble, so it is easy to measure _______________ in solution. It is used in biochemical testing to identify bacterial ____________. X-gal is split into ____________ plus the precursor for an ______________ type dye. Oxygen in the air converts the precursor to an insoluble ___________ dye that ____________ out at the location of the lacZ gene. Because of this, X-gal is used to monito ________-_____________ expression in bacterial colonies on nutrient agar plates.

beta-galactosidase, lactose, glucose, galactose, galactose, ONPG, X-gal, beta-galactosidase, o-nitrophenol, galactose, yellow, quantitatively, species, galactose, indigo, blue, precipitates, beta-galactosidase

A shuttle vector is a vector that can survive in and be moved ___________ more than one ___________ of host cell. The earliest shuttle vectors were designed to shuttle between ____________. Several components are needed to create a shuttle vector: an _________ of ________ that works in yeast, a ______________ (Cen) sequence to allow correct _____________ of the plasmid in yeast, and a gene to __________ for the plasmid in yeast, often an ___________ yeast strain that has a defect in a gene for making an ____________ organic compound.

between, type, bacteria, origin, replication, centromere, partition, select, auxotrophic, essential

T4 ligase ____________ the formation of _____________ bonds between double-stranded DNA with the 3' ___________ and 5' ____________ termini. It requires ______. It joins DNA fragments with sticky or __________ ends. It can also repair nicks in double-stranded DNA.

catalyzes, phosphodiester, hydroxyl, phosphate, blunt

A plasmid is an extrachromosomal ___________ piece of DNA that are found in various _______________ and some _____________ and are often used as cloning ____________ for genetic engineering.

circular, bacteria, eukaryotes, vectors

A gene library is a collection of ____________ segments of DNA that is _________ enough to contain at least _________ copy of every ________ from a particular _____________. The genomic DNA from the organism of interest is ___________ and __________ with a restriction enzyme, which usually has a ____________ sequence of four bases. Digestion is carried out for a __________ period that leaves many of the restriction sites ________, called a __________ digest. A suitable ___________ for the required insert size is chosen and is ________ with a __________ enzyme that produces compatible ____________ ends. The digested genomic DNA and the vector are ____________ together and transformed into the bacterial ________ cells.

cloned, big, one, gene, organism, isolated, digested, recognition, brief, uncut, partial , vector, cut, restriction, sticky, ligated, host

Expression vectors are specifically designed to place a ___________ gene under __________ of a plasmid-borne _____________. Strong promoters are used to express ____________ levels of ____________ from cloned genes.

cloned, control, promoter, high, proteins

One widely used method to check for inserts in cloning vectors uses ___________ screening. The blue/white screening uses nutrient agar and ____-_______ to produce bacterial colonies that ____________ color when an insert is ____________ within the vector. This process uses a vector that carriers the 5'-end of the __________ gene. This truncated gene encodes the ___________ _______________ of beta-galactosidase, which consists of the _____-terminal region. The MCS is located in the __________ of the lacZ alpha coding sequence. The MCS does not affect ________-______________ enzyme activity because it is ____________. However, if a ____________ segment of DNA is inserted into the MCS, the ___________ ____________ of beta-galactosidase becomes _____________ and not produced. This active form of beta-galactosidase splits ____-______, which produces a ___________ color. Plasmids without a DNA insert will produce _______-______________ and the bacterial cell that carries them will turn _________. Plasmids with an insert will be unable to make _________-_____________ and the cells will stay white. The white bacterial colony has the plasmid with the ___________, and the blue bacterial colonies have a plasmid with ______ insert.

color, X-gal, change, present, lacZ, alpha fragment, N, middle, beta-galactosidase, small, foreign, alpha fragment, disrupted, X-gal, blue, beta-galactosidase, blue, beta-galactosidase, insert, no

Another cloning method called Gibson Assembly (isothermal DNA assembly) generates _____________ single-stranded overhangs between the insert and vector, but uses a 5' ______________ to create them. The Gibson assembly reaction occurs in a single reaction __________. The fragments of DNA that are to be assembled are added ____________. For example, if the insert is very large & PCR can't be used, the insert could be assembled by ______________ multiple _________ fragments. The key is to make each PCR fragment with a 20-30bp region that has ___________ sequence as the adjacent ______ called an _____________ region. Next, the ___________ mix is added to the fragments, which contains the DNA ______________, DNA ______________, and DNA _____________. First, the ___________ degrades ______________ from each 5' end of the fragments to create a 3' single-stranded _______________ on each end. Since the fragments have overlap regions, the single-stranded overhangs are ____________, and therefore _________ at __________ temperatures (entire rxn occurs at 50 degrees). Since ___________ does not stop degrading 5' ends, there will be small single-stranded ______ which are filled in by the DNA ______________. Finally, DNA __________ seals the backbones of both strands to create one ______________ piece of DNA. Gibson assembly can ____________ multiple DNA fragments into one without adding any extra __________ pairs of DNA between insert and vector.

complementary, exonuclease, tube, first, mixing, PCR, identical, DNA, overlap, enzyme, exonuclease, polymerase, ligase, exonuclease, nucleotides, overhang, complementary, anneal, lower, exonuclease, gaps, polymerase, ligase, continuous, connect, base

Complementary DNA (cDNA) is DNA ________ of a gene that lacks _______ and therefore solely consists of the __________ sequences. Made by _______ ___________ of ___________. A cDNA library is a collection of __________ in their cDNA form, lacking ___________. cDNA is generated with the enzyme ___________ ______________. To make the library, first eukaryotic cells are __________ and ___________ is purified. The, _______ ______________ plus ________ containing oligo(dT) stretches are added. The oligo(dT) __________ the adenine in the mRNA polyA tail and acts as a ___________ for reverse transcriptase, which ___________ DNA strand complementary to the _____________. DNA ______________ is added to synthesize the opposite DNA strand, thus creating dscDNA. Linkers must be ____________ to the ends of the cDNA to allow for convenient ______________ into the cloning _______. After addition, linkers are __________ with a restriction enzyme and cDNA is ___________ into the vector. Resulting hybrid DNA molecules are then transformed into ___________, giving final cDNA library.

copy, introns, coding, reverse transcription, mRNA, genes, introns, reverse transcriptase, lysed, mRNA, reverse transcriptase, primers, hybridizes, primer, synthesizes, mRNA, polymerase, ligated, insertion, vector, digested, ligated, bacteria

In restriction enzyme cloning, The DNA fragment of interest and the vector are ______ with the same _____________ enzymes and the vector is treated with _______________ to prevent self-ligation. As a result, the ends will have _______________ single-stranded overhangs. The cut insert and vector are then mixed with _________ ___________, which _____________ links together DNA strands. The result is the _____________ of the insert and vector. If the restriction enzyme chosen generates blunt ends, ______________ is more difficult and so ____ ____________ is used because it has the ability to join blunt ends. restriction enzyme cloning is very common, and most vectors have ______________ ___________ __________ that have a series of restriction enzymes in ______________. These sites are unique and only cut the plasmid in that one ____________.

cut, restriction, phosphatase, complementary, DNA ligase, covalently, connection, ligation, T4 ligase, multiple cloning sites, tandem, position

Key Features of Cloning Vectors: The multiple cloning site is a length of DNA that contains ______ sites for ________ or __________ commonly used _____________ enzymes. It is the same as a ______________. It is a series of _____________ enzyme sites used to ____________ the DNA fragment or gene of ___________ to the ______________. These restriction enzymes sites are normally in _____________. Multiple cloning sites are designed so that the restriction enzymes sites are ___________, and the corresponding enzymes only cut the plasmid ____________.

cut, seven, eight, restriction, polylinker, restriction, connect, interest, plasmid, tandem, unique, once

Ampicillin _______________ the bacterial cell ______ structure.

degraded, wall

Gateway BP cloning reaction involves removing the gene of interest from a ____________ vector and putting it back into the ____________ vector. Involves ____________ of DNA fragment flanked by __________ and inserts DNA fragment into a vector with the __________ sequence. Only involves the ______ protein.

destination, entry, excision, attB, attP, int

Gateway LR cloning reaction involves moving the insert from the _________ vector to the _____________ vector. Involves _________ of a DNA fragment flanked by _______ and inserts the DNA fragment into a vector with the _______ sequence. Involves both the ____ and _____ proteins.

entry, destination, excision, attL, attR, int, xis

Plasmid promoters direct the __________ or __________ of the gene of interest. In ___________ operon only expresses a gene of interest under _________ conditions. Repressible promoters turn ______ gene expression.

expression, transcription, defined, off

Reporter Genes: galK The galK gene from E.coli can also be used to identify if a vector contains an insert. The galK gen encodes the protein _______________ ____________, an essential enzyme for growth on ____________. In addition, this enzyme converts ___-_____________ (2-DOG) into a _______ that kills the bacteria. The gene is primarily used in the recombineering system, so it is initially found in the ________ before the insert is added. When bacteria contain the vector without the insert, ___________ ____________allows them to grow on ____________ minimal nutrient media. In contrast, the same bacteria grow on ____-__________ because galactose kinase converts the sugar into a __________ that kills the bacteria. When recombineering replaces galK with an insert/gene of interest, then the bacteria cannot grow on _____________ minimal media, but are able to grow on ___-__________.

galactose kinase, galactose, 2-deoxygalactose, toxin, vector, galactose kinase, galactose, 2-DOG, toxin, galactose, 2-DOG

An inducible promoter is a promoter that only turns on a __________ under certain ____________, such as in the presence of a small __________ or a certain ____________.

gene, conditions, molecule, temperature

A reporter gene is a gene used in _____________ analysis because its product is convenient to ____________ or easy to ___________. Commonly used reporter genes include _________, __________, and __________.

genetic, assay, detect, lacZ, ccdB, galK

Recombinant DNA (or chimera) refers to any ____________ molecule of DNA, such as a vector plus a cloned ________, which has been merged from two different ___________ of DNA.

hybrid, gene, sources

Insertional activation is _______________ of a gene by inserting a _____________ segment of DNA into the _________ of the coding sequence. A plasmid with two __________ ____________ genes is used. One antibiotic resistance gene is used to select for the _________ cells, which have received the plasmid ___________ itself. The second is used for the ____________ and detection of _____________ DNA. The cut site for the restriction enzyme used must lie within this second antibiotic resistance gene. When cloned fragments of DNA is _____________, this antibiotic resistance gene will be ______________. Cells that receive a plasmid without an insert will be resistant to _____________ antibiotics. Those receiving plasmid with an insert will be resistant to only the ____________ antibiotic.

inactivation, foreign, middle, antibiotic resistance, host, vector, insertion, cloned, inserted, disrupted, both, first

Kanamycin ____________ mistakes during protein _____________.

induces, translation

Streptomycin __________________ the _____________ of protein _______________.

inhibits, initiation, translation

Tetracycline ___________ protein ____________ at the _________________ stage.

inhibits, translation, elongation

Chloramphenicol ____________ protein ____________ at the ______________ stage.

inhibits, translation, translocation

Gateway Cloning (2): _____ and _____ proteins recognize the attL sites of the ___________ vector with insert and induce ___________ with the _______ site of the ___________ vector. After recombination, the insert/gene of interest is now _____________ with ______ sites, and the ___________ vector sites are converted to ______. The gene product from _______ is toxic, so any bacteria that harbor the entry clone with ______ dies, and any bacteria that get the destination vector with the insert ______________. The BP cloning reaction _____________ the gene of interest from the _____________ vector and puts it back in the ___________ vector. The ____ protein recognizes the _____ sites flanking the gene of interest and induces ______________ with the ____ sites on the entry vector.

int, xis, entry, recombination, attR, destination, flanked, attB, entry, attP, ccdB, ccdB, survive, removes, destination, entry, int, attB, recombination, attP.

Molecular cloning refers to the ______________ of individual ___________ or other DNA segments and moving them into a _____________________ DNA from another organism. There are two general stages. First, the DNA region or gene of interest must be ____________ and _____________. Second, the desired piece of DNA must be _____________ into a cloning __________ that allows it to be moved into whatever final ____________ is desired.

isolation, genes, extrachromosomal, isolated, purified, inserted, vector, organism

A insert is a term used to describe the fragment of DNA that is to be ______________ to a ____________. It typically contains the ____________ of interest or other interesting DNA sequences that are being studied.

joined, plasmid, gene

Gateway Cloning (1): Another cloning method called Gateway cloning also exploits the biology of the __________ bacteriophage. Thes vectors use the integration and excision sites from the lambda ___________ for adding an ___________. It is a method of combining insert and vector that employs lambda ____ and ____ proteins and their ___________ DNA sequences (attP, attB, attL, attR) found in a series of different cloning ___________. A sequence called ______ in the lambda phage DNA combines with a DNA sequence called ____ in the E.coli genome. The _____ protein from the phage directs the ___________ event and results in the creation of _________ DNA sequences called _____ and ______ at each end of the integrated bacteriophage DNA. DNA can be excised from the bacterial genome: both the ____ and ______ proteins work together to cleanly remove only the _________ DNA, returning ____ and ____ sites to their original sequence. The first step of the Gateway system is to create the ___________ so it has two _______ sites. The insert is added to a gateway _____________ vector using ______________ ____________ ____________. Once in the entry vector, the insert is _____________ by _______ and __________ and is then competent to move into one of many different _____________ vectors in the gateway cloning system. There are a variety of ____________ vectors that have sequences needed to study the gene of __________. Moving the insert from entry vector to __________ vector uses the LR cloning reaction. The reaction uses ______ and _______ protein from lambda to ___________ the insert from the _________ vector and replace it with a gene called _______ found in the destination vector. The gene of interest replaces the ___________ gene during standard cloning using unique ___________ enzyme sites.

lambda, genome, insert, int, xis, recognition, vectors, attP, attB, int, recombination, chimeric, attL, attR, int, xis, phage, attB, attP, insert, attL, entry, restriction enzyme cloning, flanked, attL1, attL2, destination, destination, interest, destination, int, xis, excise, entry, ccdB, ccdB, restriction,

A variation of TA cloning known as TOPO-TA cloning improves the procedure by removing the __________ step. The first step is the same as in TA cloning - the insert is created by _______ with ______ DNA ____________ so it has single "___" overhangs on both ends. The vector also has "___" overhang to be compatible, but the overhang is created with _____________ 1, an enzyme that recognizes 5'(C/T)CCTT3' DNA sequences. It _____ the dsDNA vector at this sequence but does not release from the __________. Instead, the ________________ 1 becomes ______________ bonded to the 3' end of the ___________. When the 5' end of the insert nears the 3' end of the vector, the covalent bond energy is ______________ to connect the ___________ and ___________. Then the _____________ 1 is released from the DNA, leaving insert and vector linked _______________.

ligation, PCR, Taq polymerase, A, T, topoisomerase, cuts, DNA, topoisomerase, covalently, vector, transferred, vector, insert, topoisomerase, together

Plasmids can be transfected into cells transiently, meaning the plasmid is ____________ in the ___________ but does not __________ with the ___________. or it can be transfected stably, which means the plasmid ___________ into the ___________.

maintained, nucleus, integrate, chromosomes, integrates, chromosomes

A cosmid is a small __________ plasmid that carries lambda ____ sites and can carry around 45kb of _________ DNA. This allows you fill almost a whole lambda ________ with clones DNA. The cosmid is first __________ so that each end has a ______ site. In order to clone a gene of interest into the cosmid, both the gene and cosmid are _____ with the same ___________ enzyme to give identical ___________ ends (BamHI and MboI). The target DNA is often partially ____________ to allow large segments of a genome to be isolated. Ligation of the two cosmid pieces on either side of the target DNA results in a _________ of DNA with a ______ site at each end. This construct can be ___________ into lambda __________ in vitro and then used to ______________ E.coli.

multicopy, cos, cloned, particle, linearized, cos, cut, restriction, sticky, digested, length, cos, packaged, particles, infect

Key Features of Cloning Vectors: Plasmids also have terminator sequences which are positioned after the ___________ ____________ __________ (MCS). These ________ transcription of the gene of _____________. Sometimes RNA polymerase continues to make RNA through the ___________ and beyond. This is called ________-__________ and it can be reduced by having two or more ______________ in _________ on the plasmid. The terminators of eukaryotic plasmids also contain DNA sequences that add a ______ ____ ___________ onto the RNA transcript.

multiple cloning site, end, interest, terminator, read-through, terminators, tandem, poly A tail

A repressible promoter is a promoter that turns ________ the ___________ of its adjacent gene under certain _____________, such as in the presence of a small ___________ or at a certain ______________.

off, expression, conditions, molecule, temperature

In TA cloning, complementary single-stranded ___________ between the insert and vector are created by exploiting a secondary enzymatic property of ______ ___________, which as ___________ ____________ activity. This means it adds a single ____________ (dA) to the 3' ends of dsDNA. First, an insert is produced in a ___________ reaction using ____ __________ which adds a single "A" onto the ______ of the PCR product. Next, the PCR products are mixed with a vector that has _______________ 3' _______________ (T) overhang. _________ ____________ is added to connect the ___________ and ___________. This procedure is useful when convenient restriction sites are not ___________. Additionally, it does not require the researcher to _____ and ___________ the complementary vector as is done in restriction enzyme cloning.

overhangs, Taq polymerase, transferase activity, deoxyadenosine, PCR, Taq polymerase, ends, complementary, deoxythymidine, DNA ligase, vector insert, available, cut, purify

Genetic engineering via homologous recombination is known as ________________. The technique uses the enzymes for _________________ recombination from ____________ phage in order to combine an __________ and ____________. Uses lambda page enzymes (________) to connect the ends of the insert with the ______________ DNA sequence on the vector. The system of three different proteins: _______, ______, and _________ are essential for the bacteriophage to __________ its genome into its host, E. coli and they will integrate any ____________ piece of DNA into a region of __________ sequence in a vector. Recombineering has many advantages, such as: 1. the Red proteins need only a small region of ____________ in order to ____________ foreign pieces of DNA into a vector. 2. The laboratory procedure is __________ and easy. 3. The system recognizes short single-stranded oligonucleotides in addition to ___________ DNA fragments to _________ a recombination event. As a result, these can be used to create ______ deletions, insertions, or a single ____________ changes in any gene. The insert for recombineering must have approximately _____ base pairs of DNA sequence on both ends that is ____________ to the site on the vector.

recombineering, homologous, lambda, insert, vector, Red, homologous, Gam, Beta, Exo, integrate, linear, identical, homology, integrate, quick, longer, initiate, small, nucleotide, 50, homologous

Genes used in genetic analysis because their products are easy to detect are known as ______________ genes. They are often used to report on gene _______________. Commonly used reporter genes include __________, ____________, and ___________.

reporter, expression, lacZ, ccdB, galK

The tet operon confers ___________ to the antibiotic _____________ and is another example of an inducible promoter used to regulate ______________ of a foreign gene in the host bacterium. The _______ repressor protein binds to ____, an operator site in the promoter and prevents _____________ of tetracycline resistance genes. When ____________ is present, it binds to the ________ protein, releasing it from the promoter. As a result, the tet operon is induced by _______________.

resistance, tetracycline, expression, TetR, tetO, expression, tetracycline, TetR, tetracycline

Dephosphorylation is sometimes necessary to prevent _______-ligation of empty vector and is performed before _____________ (restriction enzyme cloning). Commercially available ______________ will remove the 5' ___________ group from ends of DNA since the ligation reaction requires a free 5' _____________.

self, ligation, phosphatase, phosphate, phosphate

The lacUV promoter is an extremely ______ promoters. ___________ is artificial inducer that turns on the lac promoters and stimulates ______________. ___________ binds to the LacI repressor protein, which then detaches from the __________. This allows RNA ____________ to binds. Before IPTG is added, _________ repressor prevents cloned gene from being expressed.

strong, IPTG, transcription, IPTG, DNA, LacI

The ccdB gene produces a __________ that kills its host bacteria unless the host has the corresponding _____________ gene, ______.

toxin, antitoxin, ccdA

Reporter Genes: ccdB The ccdB gene from E.coli encodes a __________ that blocks the function of DNA _________, a key enzyme for DNA __________ during bacterial growth. Bacteria with this gene ______ because they cannot _____________ their DNA. Luckily, bacteria have a gene called ccdA that produces an _____________ that inactivates the _________ and allows the bacteria to survive. If ccdA is lost or no longer _____________, the bacteria die. This reporter gene is used in the _______________ cloning system. In this system, the ccdB gene is found in the ______, and when the vector and insert are combined, the __________ gene is lost. The host bacteria that the cloning mixture is ______________ into does not have _________ in its genome, The host bacterium containing a vector without an insert __________ because the ccdB has not been replaced by the ___________. The host bacterium that gets the vector with insert ____________ because the insert replaces the __________ gene. After transformation, the only bacteria that grow have an _________, making the selection process easy.

toxin, gyrase, replication, die, replicate, antitoxin, toxin, expressed, gateway, MCS, toxin, transformed, ccdA, dies, insert, survive, ccdB, insert

Key Features of Cloning Vectors: The promoter region is found _____________ or before the site where the gene of ______________ is added. Its purpose is to direct _______ _______________ to make ___________ copies of the gene of ______________. Plasmid promoters direct the _____________ or ____________ of the gene of interest. The region includes an RNA polymerase ______________ site and any binding sites for ______________ proteins such as ____________ factors. Promoters provide the greatest _____________ among cloning vectors because they must be ________________ for their host. The promoter offers the researcher a means to ________ the expression of their gene of interest. Promoter sequences can be __________ so that the genes are only ____________ in certain growth ____________.

upstream, interest, RNA polymerase, mRNA interest, expression, transcription, binding, regulatory, transcription, variability, compatible, control, changed, expressed, conditions

Bacterial artificial chromosome (BAC) is a single copy _________ based on the ____-plasmid of E.coli that can carry very ________ inserts of DNA. Widely used in the __________ ___________ project. ____________ is necessary to transform these large constructs into E.coli host cells. Carries the second ____________ number of kb out of the three.

vector, F, long, human, genome, electroporation, highest

P1 artificial chromosome (PAC) is a single copy ___________ based on the _____-_________/plasmid of E.coli that can carry very _____________ inserts of DNA. Carries the ___________ number of kb out of the three.

vector, P1-phage, long, least

Transduction is the introduction of ____________ into mammalian cells using __________-mediated delivery.

vectors, viral

Transfection is the introduction of _________ without using ___________ into mammalian cells. Methods include cationic ________-mediated delivery, _____________ phosphate _________________, DEAE-dextran-mediated delivery, and _______________.

vectors, viruses, lipid, calcium, coprecipitation, electroporation

Huge segments of DNA, up to 2000kb or 2 million base pairs, may be carried on ____________ ____________ __________ (YACs). These are single copy ___________ based on the ___________ chromosome that can carry very long ___________ of DNA. Widely used in the ____________ ______________ project. Must have an __________ of __________ and a _____________ recognition sequence (Cen). YAC also has ______________ sequences present on both ends. For practical use, must also have a ____________ marker and a suitable ________.

yeast artificial chromosomes, vectors, yeast, inserts, human genome, origin, replication, centromere, telomere, selectable, MCS