BIOL 142 Lecture Notes - Lecture 2: Operon, Hfr Cell, Chromosome

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7 May 2018

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Bacteria reproduce by the process of Binary Fission: the chromosome in the mother cell is

replicated and a copy is allocated to each of the daughter cells.

This results in genetically identical daughter cells.

However, genetic exchanges between bacteria prior to replication of the genome result in

genetic diversity that is passed on to daughter cells. There are 3 types of genetic exchanges

that Bio 142 will touch on: 1. Transformation, 2. Transduction, and 3. Conjugation

Gene Cloning

Gene cloning is the incorporation of a foreign gene into a vector to produce a

recombinant DNA molecule that replicates and expresses the foreign gene in a

recipient cell.

•

Regulation of Gene Expression

Expression of genes is regulated by intracellular or environmental conditions.

•

An operon is a set of genes that is transcribed as a single unit and expressed

coordinately.

Specific regulation induces or represses a particular gene or operon.

○

Examples include Lac Operon

○

•

Bacterial Genome Organization

Chromosomal DNA

Plasmids

Chromosomal DNA

Bacterial chromosome is a circular molecule of DNA that functions as a self-

replicating genetic element (replicon)

•

The chromosome replicates semi conservatively; each DNA strand serves as a

template for synthesis of its complementary strand.

•

Replication of the chromosomal DNA in bacteria starts at a specific chromosomal site

called the origin and proceeds bi-directionally and the replicated chromosomes are

partitioned into each of the daughter cells.

•

Most bacteria have a haploid genome.

•

Typically, double stranded circular DNA

•

Spontaneous mutations in individual bacteria are rare

•

Plasmids

Discrete, extrachromosomal genetic elements in bacteria.

•

Plasmids replicated independently of the bacterial chromosome

•

Most plasmids are circular double stranded DNA molecules

•

Plasmids often control medically important properties of bacteria, such as resistance

to antibiotics, these are called R plasmids

•

DNA molecules that replicate as discrete gene units in bacteria are called replicons.

Replicons can be the singular chromosome or the plasmids, or both.

•

Exchange of Genetic Information

Genetic interactions between microbes allow their genomes to evolve much more rapidly

than by mutation alone

Donor DNA cannot persist in the recipient bacterium unless it is part of a replicon,

recombination between donor and recipient genomes is required to produce stable hybrids.

Recombination is most likely to occur when the donor and recipient are from the same or

closely related species.

Bacterial Transformation

Transformation is genetic alteration of a cell resulting from the direct uptake,

incorporation, and expression of exogenous genetic material from its surroundings.

•

Exogenous: produced or originating outside of an organism

•

Recombination occurs between single molecules of transforming DNA and

chromosomes of recipient.

•

Molecules of transforming DNA correspond to very small fragments of the bacterial

chromosome

•

Competence: the ability of bacteria to take up extracellular DNA and become

transformed.

•

Transduction

Bacteriophages act as vectors to introduce DNA from donor bacteria to recipient

bacteria by infection.

•

A phage is a virus that infects bacteria

•

Conjugation

Direct contact between the donor and recipient bacteria leads to establishment of a

cytoplasmic bridge(AKA Pilus) between them and transfer of part of or all the donor

genome to the recipient.

•

Donor ability is determined by specific plasmids called fertility plasmids or F

Plasmid (or F factor).

•

Bacteria that have the F plasmid are referred to as F+

Those who do not are called F-

○

•

The F plasmid can exist as extrachromosomal genetic material or be integrated in the

bacterial chromosome (via recombination)

•

When integrated into chromosome, the cell is called HFr cell (high frequency of

recombinant cell)

•

HFr cell behaves like an F+ cell, transferring F genes to a F- cell, but now it can also

donate some of the bacterial chromosome with it

•

Replication of the Hfr chromosome begins at a fixed point within the integrated

chromosome

•

Movement of bacteria usually disrupts conjugation before the entire chromosome can

be transferred.

•

Cross over events can happen between homologous genes on the Hfr fragment an the

F- DNA (recipient DNA)

•

The parts of the Hfr fragment that are NOT integrated via recombination are degraded

and lost

•

Interrupted- mating experiments

Used to determine linkage

The Hfr chromosome unwinds and is transferred to the F - cell in a linear

fashion.

○

The unwinding and transfer begin at a certain point called O or the origin

○

The farther a gene is from the origin, O, the later it is transferred to the F- cell

○

•

For Further Reading:

(most of the notes here were adapted from these sites)

https://www.ncbi.nlm.nih.gov/books/NBK7908/1.

https://www.ncbi.nlm.nih.gov/books/NBK21942/2.

Bacterial Genetics

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Document Summary

Bacteria reproduce by the process of binary fission: the chromosome in the mother cell is replicated and a copy is allocated to each of the daughter cells. However, genetic exchanges between bacteria prior to replication of the genome result in genetic diversity that is passed on to daughter cells. There are 3 types of genetic exchanges that bio 142 will touch on: 1. Gene cloning is the incorporation of a foreign gene into a vector to produce a recombinant dna molecule that replicates and expresses the foreign gene in a recipient cell. Expression of genes is regulated by intracellular or environmental conditions. An operon is a set of genes that is transcribed as a single unit and expressed coordinately. Specific regulation induces or represses a particular gene or operon. Bacterial chromosome is a circular molecule of dna that functions as a self- replicating genetic element (replicon)

Related Questions

Every year, the viral strains included in vaccinations for the flu are slightly different, based on the prevalence of the strains present the previous year. Which of the following statements is TRUE regarding the vaccinations and the treatment of viral infections like the flu?

	A.	When someone who is vaccinated encounters the virus they were vaccinated for, antigens are produced triggering destruction of the virus.
	B.	Antibodies are built up in response to viral antigens, making those with vaccines more susceptible to viral infection.
	C.	Viruses mutate quickly due to overuse of antibiotics.
	D.	Vaccinations involve introducing an antigen of a virus into the immune system, which stimulates the production of antibodies.

Recall that HIV is a retrovirus. Based on that, which of the following would NOT be an effective drug target for treatment of HIV?

	A.	Translation inhibitors
	B.	Integrase inhibitors
	C.	Reverse transcriptase inhibitors

The type of horizontal gene transfer that specifically requires a virus to transfer DNA from donor to recipient is called:

	A.	transformation.
	B.	conjugation.
	C.	transduction.
	D.	binary fission.

Binary fission differs from mitosis in that

	A.	binary fission occurs in prokaryotes, and mitosis happens in eukaryotes.
	B.	binary fission does not have a step to duplicate the genetic information, and mitosis does.
	C.	binary fission creates daughter cells that are genetically identical to the parent cell, while mitosis introduces genetic variation.
	D.	binary fission does not have a step to separate the parent cell into two daughter cells, and mitosis does.

Bacteria contain ______ in their cell walls, while Archaea contain _____ in their cell walls.

	A.	chitin; peptidoglycan
	B.	peptidoglycan; pseudopeptidoglycan
	C.	pseudopeptidoglycan; peptidoglycan
	D.	peptidoglycan; chitin
	E.	cellulose; chitin

2.Conclusion:

What have you learned from your study?

Interpret your data/ results:

Do your results support of disprove your hypothesis?

Compare and contrast your result with those of previous research.

Significance of the results: âBig Pictureâ

What is the global impact of your results?

What questions remain unanswered?

What kinds of experiments can come out of your study?

i have lab for biology and i need help with the Lab Report

the lab name is Bacterial Transformation Lab

1. Introduction:

Introduce the background and the nature of the problem your experiment is investigating.

Summarize the current knowledge of your subject.

Use examples of other research that have examined the same or a similar topic as your experiments.

Ending your introduction:

State your hypothesis

State your predictions

Give a brief statement about how you tested your hypothesis.

please help me with it is important for my lab class tomorrow

that is the lab i put the info please help me for the intro and for number 2 please be clear please

i would be happy if you help me it is due tommorrow i realy need your help on the intro and number 2 please be clear and show clear work hope you do it because it is due tomorrow

Bacterial Transformation Lab

Genetic engineering is one of the newest applications of basic knowledge in biology. It involves the isolation of specific genes from one organism and the insertion of those genes into a second organism of the same, or even different, species. Remember that a gene is a piece of DNA which codes for a product, usually a protein, which gives an organism a particular trait. The development of these techniques has generated considerable excitement and answered many questions, but it has also raised questions. Many scientists and others see genetic engineering as offering opportunities to make life better, for example, by exploring how genes are regulated, by curing diseases of genetic origin, by adding desirable traits to agricultural crops such as the ability to fix nitrogen in corn and pest resistance in cotton, and by genetically altering bacteria with genes enabling them to digest oil spills.

For almost 100 years, it has been possible to isolate DNA from cells, but only in the last 40 years or so has its function been understood. Your textbook discusses the work of such investigators as Fred Griffith and Oswald Avery, who demonstrated that if DNA was isolated from a pneumonia-causing strain of bacteria and added to cultures of nonvirulent bacteria, the recipient cells were transformed into virulent types that caused pneumonia. These transformation experiments were the first to show that genes could be artificially transferred in the laboratory. Since the time of those experiments (Avery's work was done in the 1940s), our knowledge of gene structure, function and control has increased astronomically. The application of this knowledge has created the field of genetic engineering.

Modern genetic engineering techniques depend on three critical factors:

A suitable host organism in which to insert the foreign gene;

A vector to carry foreign genes into the host; and

A means of isolating the host cells that have taken up the foreign gene.

The host organism

The most commonly used host organism is the gut bacterium Escherichia coli (E. coli). Its chromosome is a single large circular DNA molecule, called the genomic DNA, containing about 4 x 10⁶ base pairs. Not every cell will take up foreign DNA when exposed to it, but E. coli grows rapidly, dividing in less than 20 minutes, so that a single cell can give rise to a billion genetically identical descendants in a matter of 10 hours. Thus, a few transformed cells can provide a large number of offspring within a day. The strain we are using is K-12.

The vector

A vector is a DNA molecule used as a vehicle to carry genes from one organism to another. This DNA molecule can be modified by gene-splicing techniques using restriction enzymes and ligases so that it carries the genes of interest (essentially a cut-and-paste operation). Sometimes viruses are used as vectors, but for E. coli, plasmids are often used. A plasmid is a small circular DNA molecule containing 1000 to 200,000 base pairs. Plasmids exist naturally in many strains of E. coli and other bacteria. Plasmids replicate as the cell grows, independent of replication of the genomic DNA. These plasmids are transmitted to each of the product cells during cell division. Plasmids carrying genetic information that is beneficial to the host cell are maintained in a given population by natural selection. Plasmids frequently carry genes that confer antibiotic resistance, an obvious benefit.

When E. coli cells are exposed to plasmids or any other foreign DNA, some of the cells, called competent cells, will take up the DNA. We can induce cells to become competent by treating them with divalent cations, such as Ca⁺⁺, followed by a brief exposure to high temperature, called a heat shock treatment. Exactly how this treatment causes cells to become competent is not understood, but it is described as making the membranes "leaky" so that large DNA molecules (like plasmids), which would not normally pass through the cell membrane, can enter the cell. This treatment does not make all cells competent, but it greatly increases the number of competent cells. Once in a cell, the plasmids replicate and are passed to the product cells during cell division.

The selective medium

The last requirement for genetic engineering is a means of isolating host cells that have taken up the gene of interest. The techniques used depend on the growth requirements of the host organism. For example, each species of bacteria has particular nutrient requirements. The composition of the growth medium (agar or broth) can be modified to allow growth of only certain bacteria -- for example, by adding or removing nutrients or other chemicals from the medium mixture. This modified medium is called a selective medium, because it selects for the growth of only some species. By introducing an antibiotic such as ampicillin into the medium in which the bacteria are grown, you will select for growth of only those bacteria carrying the antibiotic-resistance gene on newly acquired plasmids. In a matter of days, billions of antibiotic-resistant cells, all with the plasmid, can be grown.

Genetic engineers use a modification of this technique to insert a foreign gene into E. coli. In simplified terms, the plasmid (already carrying an ampicillin resistance gene) is modified by inserting the gene of interest. The gene of interest has been isolated from another source; for example, the gene for human insulin has been isolated from the human genome. The modified plasmid, carrying the inserted gene, is then mixed with competent E. coli and the bacteria are placed in a medium containing ampicillin. Only those cells that have the plasmid will grow in the medium, and they are also the cells carrying the insulin gene (in their plasmids). These selected cells can now be used to manufacture insulin, which can be used for insulin therapy by diabetics.

The genes

In the following experiment, you will insert a plasmid into E. coli. The plasmid you will use carries the gene bla, which codes for a protein (beta-lactamase) that is secreted by the bacteria, inactivating the ampicillin in the agar and allowing for bacterial growth. The plasmid also carries an indicator gene. The indicator gene we are using was isolated from a bioluminescent jellyfish, Aequorea victoria. It codes for Green Fluorescent Protein (GFP).

The GFP gene has been modified to include a regulator system that can be used to control the expression of the gene. Gene expression in all organisms is carefully regulated to allow for adaptation to differing conditions and to prevent wasteful overproduction of unneeded proteins. The genes involved in the breakdown of different food sources are good examples of highly regulated genes. For example, the sugar arabinose is both an energy source and a carbon source for bacteria. The bacterial genes that make digestive enzymes to break down arabinose for food are not expressed when arabinose is not in the environment. But, when arabinose is present, these genes are turned on. When the arabinose runs out, the genes are turned off.

Arabinose initiates transcription of these genes by promoting the binding of RNA polymerase. In the genetically engineered pGLO DNA, some of the genes involved in the breakdown of arabinose have been replaced by the jellyfish gene GFP. When bacteria with this plasmid are grown in the presence of arabinose, the GFP gene is turned on and the bacteria glow green in UV light. When arabinose is absent form the growth medium, the GFP gene remains turned off and the colonies appear white. This is an example of the central molecular framework of biology in action: DNA ? RNA ? protein ? trait.

In summary, you will render E. coli cells competent to take up plasmid DNA. The plasmid carries two identifiable phenotypic markers:

The beta-lactamase or bla gene codes for resistance to the antibiotic ampicillin. We will use this to select for bacteria that have taken up the plasmid.

The pGLO gene codes for Green Fluorescent Protein (GFP), which is switched âONâ if arabinose is present.

Safety Procedure

For your own safety and the safety of others, it is essential that you follow all instructions about handling bacteria and disposal of materials. When you finish work for the day, swab your table with disinfectant. WASH YOUR HANDS thoroughly with soap before leaving the lab for any reason, and at the end of the day after you have cleaned your table. Report any spills to your instructor.

List of Materials

Prepared agar plates (2 LB, 2 LB/amp, 1 LB/amp/ara)

Transformation solution (50 mM CaCl₂, pH 7.4)

LB broth

1 pack of inoculation loops

Sterile transfer pipets

1 foam microtube holder w/ tubes

E. coli starter plate

1 red biohazard bag for loop disposal (common use â Loops/pipets only - NO PAPER!!!)

beigefox320

What is a genome? What is a genome made of? Describe the bacterial genome. How do bacteria reproduce? What is binary fission? Distinguish between a chromosome and chromatin. Where are chromosomes located in eukaryotic cells? What is the chromosome made of? What is a centromere? What is the centromere's role? What are sister chromatids and how do they arise? Distinguish between somatic cells and germ (gametes) cells. Describe the cell cycle. What are the different phases? What occurs during S phase? What is interphase? How is the cell cycle controlled?

Describe mitosis. What are the phases of mitosis? What occurs in each of the phases of mitosis? What is cytokinesis? How does this differ between plant and animal cells? What is a kinetochore? Where does it form? What is its role? What is the role of microtubules in mitosis? What is a centriole and what is it composed of? What is a centrosome and what is its role in mitosis? What is the metaphase plate and why is this important? What is the mitotic spindle, its role and its makeup? What defines a diploid cell? How many copies of each chromosome is in a diploid cell? What about a haploid cell? Why do the daughter cells of mitosis always have the same complement of chromosomes?

Describe meiosis. What are the phases of meiosis? What occurs in each of the phases of meiosis? Distinguish between meiosis I and meiosis II and describe how each of these processes compare to mitosis. Why do animal cells undergo meiosis? What is a gamete? What is a zygote? What are recombination, crossing-over, and chiasma? When and why does this occur? What is meant by 'homologous pair of chromosomes'? Why don't the daughter cells of meiosis have the same genetic makeup? What controls the segregation of chromosomes into the gametes?

What are genetic characters? What are genetic traits? What is an allele? What is a locus? How many alleles at each locus would a human cell have and why? Distinguish between genotype and phenotype. What is meant by a dominant gene? What is meant by a recessive gene? How do these differ? What is the genotype of a homozygous dominant organism? What is its phenotype? What is the genotype of a homozygous recessive organism? What is its phenotype? What is the genotype of a heterozygous organism? What is its phenotype? What is a genetic cross? Distinguish between the P, F1, and F2 generations. What is a true-breeding line? What is the genotype and phenotype of a cross between homozygous dominant and homozygous recessive parents? If the F1 progeny are crossed, what would be the genotype and phenotype of the F2 generation? What would be the phenotypic ratio? What would be the genotypic ratio? Be ready to generate Punnet square tests or probability analyses of crosses involving more than one genetic locus. Describe a dihybrid cross where two alleles are homozygous dominant in one parent and homozygous recessive in the other. What would be the phenotypic ratio of the F2 generation from this dihybrid cross? What is meant by independent assortment? Be prepared to calculate probabilities resulting from different crosses.

Distinguish between complete and incomplete dominance. How would incomplete dominance alter the results of a hybrid cross? What would the resulting F2 generation phenotype and genotype ratios be? What is codominance? How does this affect the results of crosses? Be prepared to determine likely blood types of parents based on the blood types of their offspring. What is a polygenic trait? What is a carrier state for a genetic disease? What is meant by linked genes? How does this affect expected outcomes of genetic crosses? How is gene linkage used for chromosomal mapping? Distinguish between autosomes and sex chromosomes. What sex chromosomes are found in women? What sex chromosomes are found in men? What are sex-linked traits? Be prepared to give the results of crosses that involve sex-linked recessive traits. What is the process of nondisjunction? What are the likely results? Why is this important? What is a Barr body? Recognize the genetic abnormality of Downs Syndrome, Turner's Syndrome, and Klinefelter's Syndrome.

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BIOL 142 Lecture Notes - Lecture 2: Operon, Hfr Cell, Chromosome

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