Define the term biotechnology and describe what fields it pertains to.
Biotechnology: The science of using living systems to benefit humankind.
Biotechnology can be used for industrial, medical, and agricultural applications with synthetic biology (assembling D N A pieces into new genetic elements)
Define recombinant DNA technology (DNA cloning) and explain the example of using microorganism to make human insulin or the example in the biotechnology worksheet of cloning an anti-microbial fungal gene.
Recombinant DNA technology (also called DNA cloning): a piece of DNA is copied from one organism and pasted into a small piece of DNA called a plasmid.
The insulin gene from humans was inserted into a plasmid. This recombinant DNA plasmid was then inserted into bacteria. As a result, these transgenic microbes are able to produce and secrete human insulin. Many prokaryotes are able to acquire foreign DNA and incorporate functional genes into their own genome through “mating” with other cells (conjugation), viral infection (transduction), and taking up DNA from the environment (transformation).
Explain the steps of creating recombinant DNA (DNA cloning).
- PCR makes multiple copies of GeneX
THe polymerase chain reaction (PCR) is DNA replication invitro, multiplying segments of target DNA(a few kbp in length) up to the billionfold during amplification.
PCR requires the following components which are mixed in a tube:
Template DNA—contains the target DNA to be amplified
Primers—short pieces of DNA that designate where the copying will begin and end
DNA polymerase—enzyme that will replicate the DNA
Buffer solution—provides the proper pH and salt concentration for the replication reaction to occur efficiently
Steps in PCR: - Denaturation– separate strands of template D N A by heating at a high temperature
- Annealing: lower temperature to allow primers to bind to target sequence
- Extension: D N A polymerase extends primers using original D N A template
- Restriction enzymes cut the PCR products and plasmid
- Ligate the plasmid and PCR products together
- Transform the cell
Explain the purpose of PCR (polymerase chain reaction) and describe what happens in each step.
The P C R amplifies specific D N A sequences.
(a) Target D N A is heated to separate the strands, and a large excess of two oligonucleotide primers, one complementary to each strand, is added along with D N A polymerase.
(b) Following primer annealing, primer extension yields a copy of the original double-stranded D N A.
(c) Two additional P C R cycles yield four and eight copies, respectively, of the original D N A sequence.
(d) Effect of running 20 P C R cycles on a D N A preparation originally containing 1 copy of a target gene. Note that the plot is semilogarithmic.
Describe restriction endonucleases, where they cut DNA (restriction sites), and how they cut (leave sticky ends).
Restriction endonucleases (restriction enzymes) are used to cut the DNA at specific sequences called restriction sites.
Why might bacteria have these naturally?
Protect the bacteria from invading bacteriophages (viruses)
- DNA fragments separated by gel electrophoresis
Explain the job of ligase and what DNA pieces it can glue together.
Any DNA molecules cut with same restriction enzyme can be joined together by another enzyme called ligase because of sticky ends.
Explain the purpose of gel electrophoresis, how it works, including what the charge of the DNA molecule is and the positioning of the electrodes.
Agarose gel acts like a maze of small holes
Small DNA fragments move quickly through the small pores
Larger fragments take more time to “wiggle” though.
DNA (negatively charged) moves through gel toward the positive end by adding an electrical current
Define the term plasmid vector and describe the 3 essential features of plasmid vectors.
Plasmid vector: Carries the inserted piece of DNA into the bacterium where it gets replicated/expressed
Plasmid vectors usually contain:
1. Ori (origin of replication)
2. Antibiotic resistance markers, usually at least two
3. Known restriction sites often in the form of several grouped together called a multiple-cloning site or polylinker.
Explain the three different types of horizontal gene transfer: transformation, transduction, and conjugation.
There are 3 different methods:
- Transformation: competent cells take up DNA directly from their environment
- Transduction: DNA transferred by bacteriophage (virus that infects bacteria)
- Conjugation: One live bacterium transfers the plasmid to another live bacterium via a pilus.
For transformation: definite the term competent cells, understand the difference between artificial and natural competence, describe how the DNA gets inside the cell.
Transformation is when competent cells take up DNA from the environment.
Cells are either naturally competent or artificially competent
- Natural: Competence factors: surface proteins that allow bacteria
- Artificial: treatments can increase membrane permeability (CaCl2, electroporation )
Large (7,00-10,000 bp) DNA fragments can be accepted.
- Donor DNA associates with DNA binding protein on the cell surface
- One strand of the DNA is transported into the cell while the other strand is hydrolyzed by a nuclease.
- At least part of the donor DNA can move into the cell’s chromosome by homologous recombination
For transduction, describe the difference between virulent viruses that use the lytic cycle and temperate viruses that use the lysogenic cycle and lytic cycle. Explain the difference between general transduction and specific transduction.
Transduction: using a virus that infects bacteria to pass genes from one bacteria to another.
Viruses can infect using to different approaches
- Virulent viruses infect using the lytic cycle
- Temperate viruses infect using the lysogenic cycle
- A bacteriophage injects its DNA into a bacterium
- During the lytic cycle of virulent phage, the bacteriophage takes over the cell, reproduces new phages, and destroys the cell - Phage DNA is replicated, and the bacterium’s chromosome is broken down. Viral proteins are synthesized
- When new phage particles are assembled, some phage heads may get stuffed with bacterial rather than viral DNA
- If that phage infects another bacterium, it can transfer genes from the lysed bacterium into a new cell.
Temperate phage can incorporate into the cell’s chromosome (lysogenic) but can be induced into a lytic cycle in which many infective phage particles are produced.
Transduction occurs when a bacteriophage transfers bacterial DNA from one bacterium to another during sequential infections.
There are two main types:
- Generalized transduction: when the virus picks up a random piece of bacterial chromosome by mistake during the lytic cycle.
- Specialized transduction: occurs at the end of the lysogenic cycle, when the prophage is excised, and the bacteriophage enters the lytic cycle.
For conjugation, describe the process step by step.
Conjugation: when DNA is transferred between 2 liver bacteria.
- DNA is transferred between cells
- Requires physical contact between cells
- F plasmids; code for pilus
Describe the purpose and process of using antibiotic resistance genes for selection.
We use antibiotic resistance and a reporter gene.
Each plasmid has an antibiotic resistance gene, so if we grow the bacteria on media containing that antibiotic, only bacteria that took up the plasmid will live.
Explain the concept of using a reporter gene (blue-white screening) to check for a gene insert in a plasmid.
A reporter gene is another gene sequence artificially engineered into the plasmid that encodes a protein that allows for visualization of DNA insertion.
Blue/white screening
- Insertional inactivation of gene within lacZ (encodes the β-galactosidase enzyme) used to detect cloned D N A
- Transformants are plated on media containing ampicillin and X-gal which turns blue when broken down by β-galactosidase.
- Cells containing vector with insert are white because no β-galactosidase formed.
Explain the purpose of the CRISPR-Cas9 in bacteria.
Genome editing: Use of C R I S P R/Cas9 system from Streptococcus pyogenes to alter eukaryotic genomes in living cells
Sequence Targeting by the Cas9 Protein
- Cas proteins of C R I S P R systems function as endonucleases when guided to nucleic acids by binding of C R I S P R R N A s (c r R N A s)
- Synthetic R N A (synthetic guide R N A [s g R N A]) can be designed to recruit Streptococcus Cas9 and bind to target D N A, enabling cutting in genome of almost any cell
- At cut site, D N A can be ligated or used to insert new D N A
Sequence Targeting by the Cas9 Protein
. Also requires protospacer adjacent motif (P A M) on target D N A for complete endonuclease activity
- Various methods of C R I P S R system delivery by injection
. Plasmid
. s g R N A and mR N A can be made in vitro
- Homologous recombination can be used to incorporate new D N A (insertion)
- Nonhomologous double-stranded D N A break repair pathway can ligate after deletion
Describe in general terms how CRISPR-Cas9 can be used for genome editing.
C R I S P R Editing in Practice:
- Edit genomes of crops and farm animals
- Edit the human genome to treat genetic diseases
- Treat other diseases (for example, viral diseases)
- Diagnostic tool to detect pathogens
Ethical questions about use in humans:
https://www.npr.org/sections/health-shots/2023/03/06/1158705095/genome-summit-gene-editing-ethics-crspr
Explain the fact the viruses are acellular, obligate, intracellular parasites.
acellular: not made of cells
Obligate intracellular parasites:
- Lack genes needed for successful reproduction
- Require a host cell to reproduce
obligate intracellular pathogen microorganism that cannot synthesize its own ATP and, therefore, must rely on a host cell for energy; behaves like a parasite when inside a host cell, but is metabolically inactive outside of a host cell
- Infectious, acellular pathogens
- Obligate intracellular parasites
- Lack genes needed for successful reproduction
- Require a host cell to reproduce - DNA or RNA genome
- Genetic material is surrounded by a capsid (protein coat)
Define the term virion.
virion: inert particle that is the reproductive form of a virus
Describe what viruses are made of: a genome (RNA or DNA), a capsid, and some have an envelope, including its composition.
All viruses have genetic material and a capsid.
- Capsid(protective coat): made of capsomeres protein subunits
- Nucleic acid (DNA or RNA)
Some viruses have an envelope (enveloped) and others do not (naked or non-enveloped).
- Envelope (phospholipid membrane)
- Spike proteins (glycoproteins that help cell entry)
Explain the concept of host range and why viruses normally have a limited host range.
Most viruses can only infect one kind of cells or a few species = HOST RANGE
- Having a wide host range is rare.
- Viruses that infect bacteria are called bacteriophages.
Why is host range so limited?
- Cells must have specific receptor sites that the virus can attach to
- Cell must contain the enzymes the virus needs to uncoat
- Cell must have the rest of the synthetic machinery
Explain the concept of transmission, including what a vector is, both mechanical and biological.
Transmission: how a virus is passed from host to host.
Viruses can be passed by
1. Direct contact
2. Indirect contact through an object
3. Vector: which is an animal that passes the virus from one host to another
- Mechanical vector: virus travels outside vector
- Biological vector: virus travels inside
Describe the three most common capsid shapes.
Viruses can vary in the shape of their capsids
The shape of the capsid is determined by their capsomeres.
Helical: rod-shaped
Polyhedral: many sided
Complex: have features of both
Identify the two main groups that classify viruses.
Viruses are not classified under the three domains of life, but still need classification.
However, they can mutate so quickly that it’s difficult to label them with a genus and species.
There are two main systems of classification:
- International Committee on Taxonomy of Viruses (ICTV) classifies viruses into families and genera based on viral genetics, chemistry, morphology, and mechanism of multiplication.
- The Baltimore System classifies viruses according to their genomes.
Know all the different types of genomes that viruses can have and explain what they mean (ssDNA, dsDNA, ssRNA + strand, ssRNA – strand, dsRNA, and RNA with reverse transcriptase.
[Genome, family, example virus, clinical features]
dsDNA; enveloped:
(Poxviridae, Orthopoxvirus, skin papules, pustules, and lesions), (Poxviridae, Parapoxvirus, skin lesions), and (Herpesviridae, Simplexvirus, Cold sores, genital herpes, sexually transmitted disease)
dsDNA, naked:
(Adenoviridae, Atadenovirus, Respiratory infection (common cold)), (Papillomaviridae, Papillomavirus, Genital warts, cervical, vulvar, or vaginal cancer), and (Reoviridae, Reovirus, Gastroenteritis severe diarrhea (stomach flu))
ssDNA, naked:
(Parvoviridae, Adeno-associated,dependoparvovirus A, Respiratory tract infection), (Parvoviridae, Adeno-associated dependoparvovirus B, Respiratory tract infection)
dsRNA, naked: (Reoviridae, Rotavirus, Gastroenteritis)
+ssRNA, naked:
(Picornaviridae, Enterovirus C, Poliomyelitis), (Picornaviridae, Rhinovirus, Upper respiratory tract infection (common cold)), and (Picornaviridae, Hepatovirus, Hepatitis)
+ssRNA, enveloped: (Togaviridae, Alphavirus, Encephalitis, hemorrhagic fever), (Togaviridae, Rubivirus, Rubella), and (Retroviridae, Lentivirus, Acquired immune deficiency syndrome (AIDS))
−ssRNA, enveloped:
(Filoviridae, Zaire Ebolavirus, Hemorrhagic fever), (Orthomyxoviridae, Influenzavirus A, B, C, Flu), and (Rhabdoviridae, Lyssavirus, Rabies)
