Recom final exam
CRISPR has a 75-85% success rate of targeting the correct gene. What is one way this success rate could be increased?
the modified cas9 enzyme, nicks, makes the crisp complex more specific because it only has one "scissor" mechanism, therefore requiring the use of 2 guide RNAs. Cas 9 Nickase mutants introduce targeted single-strand breaks instead of double stranded breaks. ****2 guide RNAs, so statistically insertion will happen in the RIGHT PLACE which we need for something like cystic fibrosis ex: prevent mosquitos from being able to pass on malaria=GENE DRIVE, pest control -were talking about releasing this into the wild, would I be comfortable with genetically engineering mice and will pass this on so that we can't be spreading lyme disease?
Explain how only a few SNPs are sufficient to distinguish 4 different fish species
the species are different enough that only a few SNPs are needed to determine whether you are looking at DNA from cod or hake or herring. these SNPs cannot be quickly-mutating either- they need to be stable in the fish population.
What effects does the toxin (in the plant) have on the insects?
toxin binds to gut epithelium, perforation of gut membrane, spores germinate and proliferate
steps of pcr
1.) Denature - Hydrogen bonds between DNA complementary strands broken @95°C 2.) Anneal - Primers attach to DNA templates @55-65°C 3.) Extend - Taq Pol is able to operate and lengthen the DNA @72°C
gene editing conclusions
need more research on benefits and risks irresponsible to use gene editing until relevant safety and efficacy issues are resolved, and until societal consensus is reached about appropriateness -human genome is shared among all nations, similarly our PLANET IS -pollution alters our genome just as much as this
differentiate between non-GMO project and USDA organic labels
non-gmo project-take food sample, work with for profit labs, PCR based, targeted for "relevant GM events" -non-profit, its propaganda, not gov. regulated -doesn't mean anything USDA organic-not just non-GMO, reduced pesticides/absence of herbicides/chemicals, also not GMO
When DNA is introduced into plants using agrobacterium, is it specific or random in integration?
random in the plant genome
ex of synthetic bio
synthetic genome of M. mycoses on computer-took out DNA and inserted completely synthetic genome using TAG stop codon to recognize expand genetic code to create new proteins, make protein based medications, cells could replicate
Restaurants sometimes substitute fancier fish with cheap fish to charge more. how can this be regulated?
use SNP chip for quality control of restaurant suppliers or kitchens.
make informed argument about CAR-T cell therapy use in a clinical setting
we're in desperate need of effective treatments for cancer, taking advantage of body's own natural defenses, the immune system. chimeric antigen receptors-CAR-T cells. for blood cancers because there are antibodies that can recognize them that are not expressed on other tissues.
given bacteria with the following configuration of the lac operon, explain what you expect w and without lactose I+OCZ-Y+/I-O+Z+Y-
without-permease with-permeate and B-gal
Argue for or against edible vaccines
would all banana get modified? would it be possible to keep GM and non-GM bananas separate? Whether communities would trust a vaccine that is not from a needle and a doctor? wether this is an ethical use of genetic engineering. is anyone allergic to bananas? needs to be a blue banana, only at medical centers, not sold in stores so that the amount of bananas is regulated. it should be like any other treatment modification--> also it should be a very small banana so that there is not a ton left over. it should be something that can be stored for a very long time, so maybe not a banana, unless you can freeze it and put it in a smoothie. its just like transitioning from a pill to a liquid medication.. needs to be regulated, and need to have no waste from it (peel could still contain vaccine)
describe the emerging field of synthetic biology
"Synthetic biology is a) the design and construction of new biological parts, devices and systems and b) the re-design of existing natural biological systems for useful purposes." ex: alter acorn to build into house
describe CRISPR/Cas9 system components
-changing what's already in genome-to make something not disease causing and other reasons 1. guide RNA to find sequence and bring custom sequence 2. Cas 9 protein cuts the double strand=break 2 strategies 1. non homologous-end joining-can often lead to changes in base pairs, but cells are going to try to fix it so they will delete from the broken sides, creates deletions, breaks protein usually ex: deletion 2. homology directed repair-involves insertion of DNA, 2 double stranded cuts Cas-9 gene-75-85% of time induces gene it wants to -25% affects other genes ****bacteria USE THIS SYSTEM TO PROTECT AGAINST VIRAL DNA, why it exists in real world creating dsb=a problem
choose and describe the appropriate molecular genetic tools that enable a gene of interest to be identified, amplified, and studied in an organism
???
After the Cas9 enzyme catalyzes its reaction, how is a mutation created?
A double stranded break is fixed in 2 says: homologous recombination-where a piece of homologous DNA is inserted into the break, binding together the 2 pieces of DNA, or non-homologous end joining, where ends of the cleaved DNA are chopped back by the cellular machinery, and then the ends are joined together, resulting in random deletion of bases at the cut site.
Describe what Agrobacteria are and how they can be useful for genetic engineering. What type of organisms can they be used to engineer?
Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering. The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation.[13][14] The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. Transformation with Agrobacterium can be achieved in two ways. Protoplasts or alternatively leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral dip method:[17] inflorescence are dipped in a suspension of Agrobacterium, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance (or another marker of interest), and plants that have not integrated the plasmid DNA will die when exposed to the correct condition of antibiotic.[15] Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun. Agrobacterium is listed as being the vector of genetic material that was transferred to these USA GMOs: Soybean Cotton Corn Sugar Beet Alfalfa Wheat Rapeseed Oil (Canola) Creeping bentgrass (for animal feed) Rice (Golden Rice)
How does a typical bacterial gene differ from a eukaryotic gene? list 2 features that differ
Bacterial genes have no introns and eukaryotes do. Bacteria have operons, or strings of co-regulated genes while eukaryotes (mostly) do not. Eukaryotes have enormous complicated promoters with many different consensus sequences; bacteria have consensus sequences within their promoters, but they're much shorter and less complex promoters.
discuss biological and ethics challenges of synthetic biology
Bioterrorism The ability of synthetic biology to produce known, modified or new microorganisms designed to be hostile to humans is a major concern, and has been demonstrated by the synthesis of the polio virus and the pandemic Spanish Flu virus of 1918 The drive to create a microorganism that can turn biomass into fuels such as ethanol or hydrogen is a major focus of research, which has prompted a concern that patenting may lead to the creation of commercial monopolies or inhibit basic research. no local production of natural Artemisia could be sustained in developing countries, thereby maintaining the discrepancy of wealth and health between rich and poor nations. creating artificial life
How exactly would you create a GMO marijuana plant with multiple copies of THX-synthase? [3 points] What is your opinion on the use of GMO marijuana plants? [2 points]
GMO???? I thought GMO is only selective breeding -cross breed, but if they don't exist you can add in more THX-synthase could use CRISPR to make deletion so now we are creating plants with the ability to make THC, but are making other metabolites for treating cancer genetic engineering
Comparing STRs or SNPs to distinguish individuals is assuming the genetic markers occur at constant frequencies across the population. Is this the case for using gene expression differences to distinguish individuals?
Gene expression can change within an individual during its lifetime or because of its environment- if gene expression can change within an individual, it won't be a stable marker of a population or species of fish, the way the genome is
How do gene therapies aid in genetic disorders/illnesses?
Gene therapies aid in genetic disorders/illnesses by introducing genetic material into specific cells to compensate for abnormal genes or make a functioning protein when faulty or missing. A correct copy of the gene is inserted into cells using a virus as a vector to deliver the new gene into infected cells.
Researchers halted gene therapy trial when they detected an altered gene in semen of men. Why?
Gene therapy does not cause heritable changes to the genome, this is one of the reasons its a possibility in humans-we can treat disorders without changing allele frequencies within the human population. CRISPR and other genome editing strategies are much more controversial because they do cause heritable changes in the genome. this trial would be halted because the therapy was causing changes in the gametes
compare and contrast synthetic biology and GMOs
Genetic modification refers to a range of methods (such as selection, hybridization, and induced mutation) used to alter the genetic composition of domesticated plants and animals to achieve a desired result synthetic biology involves bringing engineering principles to biotechnology. It's an approach meant, ultimately, to make it easier for scientists to design, test, and build living parts and systems—even entire genomes.synthetic biology is about writing and programming new DNA with two main goals: create genetic machines from scratch and gain new insights about how life works.
What are the differences between genome editing, genetic engineering, and genetic modification?
Genome editing makes changes to what is already existing in the genome. Genetic engineering is a permanent change to DNA, target embryo/fertilized egg, target embryonic stem cell. Genetic modification can be more imprecise in gene insertion and is usually done by controlling the breeding and reproduction of organism (dog breeds triploid bananas)
scientific argument for or against genome editing
Genome editing offers a greater degree of control and precision in how specific DNA sequences are changed. It could be used in basic science, for human health, or improvements to crops. There are a variety of techniques but clustered regularly inter-spaced short palindromic repeats, or CRISPR, is perhaps the foremost. only use in lifesaving circumstances and only if healthcare is universal
in vivo in vitro
PCR is considered to be "in vitro." "In vitro" refers to a process performed outside of a living organism (i.e. in a test tube), as opposed to "in vivo," which refers to processes performed within a living organism. The main "in vivo" form of DNA amplification is referred to as cloning, where the amplification takes place within a living organism (i.e. transformation of E. coli with plasmid DNA).
How can you design a screening method to detect the cystic fibrosis mutation?
PCR test. Design primers that flank the mutation, then run a gel (with controls) to see how large the PCR product is. You could even design primers where one of the primers ends within the deletion region-so then if the persons if the person's DNA has a deletion, you will not have extension of the PCT amplicon, and you will see no product amplify. Wool need a positive control reaction to make sure PCR is possible, and the reason no amplification occurred is because there's a deletion. of course you could also do Sanger sequencing.
Non-GMO Project (a bit of a scam) uses a PCR-based method to verify that various products do not contain genetic modifications. You land a job in the lab of the Non-GMO Project after graduation, and your first task is to test a bag of Doritos for genetic modifications, but your boss refuses to give you instructions. Luckily, you took Bio 226 this semester. Outline what steps you would follow to check for genetic modifications (i.e. how to run PCR, how to test and see if the genome is genetically modified...)
PROCEDURE 1. Extraction of DNA From Food Samples Label one screw-cap tube "non-GMO" and the other "test". 2. Setting Up PCR Reactions add 20 µL of the primer indicated on Table 1 to each PCR tube. Cap tubes. Using a fresh tip for each tube, add 20 µL of the DNA sample indicated on Table 1 to each PCR tube, Place PCR tubes in thermal cycler and program the cycler for: Initial Denature: 1 cycle at 94 °C for 2 min. Amplification: 40 cycles at 94 °C for 1 min (denature), 59 °C for 1 min (annealing), and 72 °C for 2 min (extension). Final Extension: 1 cycle at 72 °C for 10 min. Hold: 4 °C indefinitely. 3. 3% Agarose Gel Preparation 4. Electrophoresis of PCR Products RESULTS After destaining, gels can be analyzed by looking at test food lanes (Table 3) to determine if the DNA bands for the 35S promoter and NOS terminator genes are present in the known locations on the gel. The presence or absence of a 200 bp band in lane 5 indicates whether or not the test food contains GMOs. APPLICATIONS AND SUMMARY Polymerase Chain Reaction (PCR) is used to amplify DNA, allowing for a wide range of DNA lab testing. One area of testing now possible with PCR is to identify GMOs by testing for presence or absence of the DNA sequences used in the genetic modification of food crops. Typically, a crop is genetically modified to confer an advantage against natural deterrents to ideal yields, e.g. pests (Figure 3), diseases, drought conditions (Figure 4), etc. Because the advantage is gained by inserting genetic material from a different species into the crop plant's own DNA, potential human health and environmental risks have been identified with the use of GMOs. One environmental concern is the ability of the genetically modified DNA to be exchanged unintentionally through pollination processes, which could lead to genetically modified DNA entering the genomes of crops intended to be sold as non-GMOs.
In personal opinion, knowing the risks, would you support taking on gene therapies, if you're an insurer, to help many suffering from genetic illnesses? why?
Pros -very large risk of insurers to support therapies that don't have enough data on -help people affected by debilitating diseases Cons -many people affected don't have access to the therapies because financially can't afford it -what incentives do researches have if they can't implement the therapies they make and don't get to make differences in health
How does a DNA analysis of STRs differ from SNPs?
STRs are based on repeats, while SNPs are single-base changes. STRs would be examined through PCR and gel electrophoresis. SNPs could be identified using a chip with many probes, where each probe binds to one specific SNP mutation or the other.
How does Sanger sequencing differ from Next-Gen sequencing?
Sanger sequencing works on one sequence at a time, using a gene specific primer to start extension. NGS works in massive parallel, starting extension of its many sequences using a single primer that matches to adapter sequences that are added on the ends of the sequences of interest. Sanger visualizes the DNA sequence by running labeled chain-terminated fragments through a gel, while NGS uses a computer-based camera to watch the addition of transiently-labeled nucleotides to the growing DNA chain.
What are some risks that insurance companies want to avoid when it comes to supporting newly developed gene therapies?
Some risks are that newly developed gene therapies are built and established on new methods and practices that have not been fully adopted or used throughout the research world. Such can pose issues in efficacy that were unknown before. Furthermore, the gene therapies haven't been around enough to understand if their effects last.
How can you evaluate whether your insert DNA integrated where you hoped it would?
Southern blot (DNA) and a PCR. The PCR primers can be designed properly where one primer is in transgene and another one in the expected flanking site. The product can be sequenced to ensure the correct product. ???? fluorescent in situ hybridization (FISH) will give you an answer, and tell you things like whether you are too close to the centromere or not. If you want a more specific location and to get information on copy number as well, then whole genome seq is the way to go. If that is too costly, then you could do inverse PCR, but you will likely have to modify this approach if you have multiple insertion sites. I would suggest using a restriction digest that you know is a single cutter within your transgene sequence, thus hopefully minimizing any overly long templates that could arise from tandem repeats. But you are still going to have to do Solexa sequencing, or make a custom chip, or find some other way to get and analyze single reads (unless you get lucky and have only one insertion site) ***just use PCR
explain" standardization, modularity, and abstraction to distinguish synthetic biology from previous recom DNA approaches
Standardization involves establishing definitions of biological functions and methods for identifying biological parts, as with the registry of standard biological parts Modularity is used in other engineering disciplines to insulate interacting systems from each other and render them interchangeable. Abstraction includes establishing hierarchies of devices and modules that allow separation and limited exchange of information between levels, and developing redesigned and simplified devices and modules, as well as libraries of parts with compatible interfaces
How does synthetic biology differ from classic genetic engineering? List how they are similar and different/
Synthetic biology is different from genetic engineering because synbio uses engineering principles to achieve system-wide changes or the introduction of an entire biochemical pathway-synbio is bigger than genetic engineering. Synthetic biology and genetic engineering are similar because they both end up modifying the genomes of existing organisms
difference between plant and animal
plant-cell wall-annoying totiplant-leaf regrow new plant animal-no cell wall stem cells, embryo HR-known insertion point why can't we make new human from stem cells? :/ ??? why can't plants do homologous recombination? we don't know
What is the purpose of the refuge area in crops?
refuge area is a safe area for insects, no pesticides on or in plants -allows non-resistant bugs to survive, and breed with resistant ones keeps resistant genes low in population
standardization in synthetic bio
standard products, she thing everywhere, DNA from all sources can go together
3 principles of synthetic biology
standardization, modularity, abstraction
abstraction in synthetic biology
--> o ---> abstract thinking ex: a promoter, a ribosome binding site, and the coding region go a gene are all needed for any transgene -think abstractly (detail-free) about what's needed to express a transgene
diagram steps needed to create a transgenic plant or animal
1. Isolate DNA that codes for the protein you want to express. This protein could be anything! In this case we want to express a protein that is found in most normal plants, but isn't made in our mutant plant because of an insertion in the DNA (basically, the cells of the plant can't read the instructions and make the protein because the insertion is in the way). Therefore, we will use a piece of DNA from a normal plant that has the correct set of instructions. Transgenic plant step 2 2. Insert the DNA into a plasmid. A plasmid is a piece of DNA that can replicate on its own. Bacteria are used to plasmids and will readily accept them even if they contain foreign DNA. Transgenic plant step 3 3. Insert the plasmid into bacteria. Grow a large amount of bacteria containing this plasmid. This isn't just any bacteria! The type of bacteria that researchers use for creating transgenic plants is called Agrobacterium tumefaciens (more on this below!) Transgenic plant step 4 4. Dip the flowering plant into a large amount of bacteria. It is important to wait until plants are flowering to dip them because in order to grow new plants with this piece of DNA it is necessary to insert the DNA into the cells that make new plants - the sex cells! Specifically, we want the bacteria to insert DNA into egg cells found at the base of flowers. Remember - in this case we are dipping our mutant dying plant since we want to see if the instructions help "fix" the dying phenotype. Transgenic plant step 5 5. Give bacteria the opportunity to insert the DNA into the plant cells. Agrobacterium tumefaciens has the natural ability to insert new DNA into a plant's genome. Click here to read more about how researchers have learned to utilize this natural phenomenon! Transgenic plant step 6 6. Select for plants that have the insertion. The agrobacteria insert this new DNA into a huge number of cells. Some of these cells (the egg cells, specifically) are the cells that form seeds which will grow into new plants. Therefore, it is this next generation of plants that may contain the insertion! Not all of the offspring will have this insertion, however. In order to only continue studying the plants with the insertion, an extra piece of DNA is included in the plasmid used in the second step. This extra DNA encodes for resistance to a specific antibiotic. Basically, a huge number of seeds will be sown out on either soil or agarose that contains a specific antibiotic and only the seeds that have the DNA encoding resistance to this particular antibiotic (and therefore the seeds that also contain the DNA that we are interested in) will grow! ******callus of tiny plant tissue, select using antibiotics, callus is undifferentiated-can change the hormones so that it creates leaves and. humans are limited for humans and mammals-have to do embryonic cells.
how to make a transgenic pig
1. The following steps may be used to create a transgenic pig that secretes the bacterial enzyme in its saliva, and therefore excretes less polluting feces. 1. First, one may order the promoter region (Mus musculus strain C57BL/6J) in a plasmid with the appA phytase Escherichia coli 042 downstream from it in the same plasmid. Because the bases in these regions are already known, GenScript can synthetically create the bases in these sequences and put them together in a plasmid with specific cut sites on each side. For example, an EcoRI cut site could be placed upstream of the promoter region and a HindIII cut site will be placed downstream of the E. Coli region. 2. When the plasmid arrives with the incorporated regions, a digest could be done with EcoRI and HindIII to move around the fragments, but that is not necessary for creating the transgenic pig. 3. PCR may be used to amplify the entire plasmid. 4. The amplified plasmid containing the promoter and necessary phytase E. Coli will be inserted into the nucleus in each of 100 pig embryos using the microinjection technique under an inverted microscope. Because recombination does not always take place, the promoter region for the parotid secretory protein will only be successful a small fraction of the time. 5. As the embryos develop and are eventually born and grow into piglets, DNA samples may be taken to genotype the pig. Known primers may be used to amplify the promoter (Mus musculus strain C57BL/6J) and appA phytase E. Coli region to see if recombination, transformation, and translation were successful. As an alternative, each pig's feces could be tested for levels of phosphorus, but DNA analysis may be preferable. 6. Let's say you amplify each pig's DNA and find that only 10% of the pigs contain the appA phytase E. coli and promoter region, so only 10% of the pigs are secreting the necessary bacterial enzyme in its saliva to excrete less polluting feces. You're not satisfied and decide to try another method. In a next experiment, you may try using the CRISPR/Cas 9 system to edit the pig embryo genome so that it encodes appA phytase Escherichia coli 042 and the promoter region (Mus musculus strain C57BL/6J). The same procedure of taking a DNA sample and amplifying it after the pig has developed can be used to confirm success. need to insert embryo into mother. let develop and then insert=could get chimeric pig, must wait one more generation
Fix a fully gene using CRISPR technology
1. a guide RNA that is complementary to the target sequence must be constructed, which is then inserted into the Cas 9 enzyme, which creates the CRISPR complex. 2. The complex is inserted into the cell, and once the good RNA finds the target DNA sequence, was 9 makes a double stranded break. 3. The target gene is now disabled and can be "fixed" by adding a new DNA sequence.
List and explain the purpose of 3 basic components required for a bacterial cloning vector (ex: plasmid)
1. a selectable marker ex: antibiotic resistance will allow you to kill off bacteria not carrying your plasmid 2. an origin of replication will allow the plasmid to be replicated in all cells 3. a multiple cloning site allows the insertion of any genes of interest into the plasmid sequence
how crispr works
1. construct an RNA guide that includes a part matching the desired DNA sequence 2. attach the RNA guide to an all-purpose Cas9 cutting protein, creating the CRISPR tool. 3. Introduce the CRISPR tool into the cell of interest. The guide RNA finds its DNA match in the genome. 4. The Cas9 protein cuts both strands of the DNA in a gene so that the gene will be disabled or, with the insertion of a segment of engineered DNA, modified.
know from dog olfactory receptor PCR project
1. how to isolate genomic dog DNA 2. design primers to amplify CfOR0432 3. create primer stocks of the appropriate concentration for use in PCR, given the moles of DNA 4. run PCR 5. interpret gel of PCR products 6. extract and purify gel DNA 7. Find concentration of DNA using Nanodrop machine 8. prepare samples for sanger sequencing 9. analyze dog olfactory receptor sequence chromatograms
explain or draw the process of plant tissue culture to generate genetically engineered plants
3 options for introducing new bacteria into plants: 1. biolistics-gun 2. electroporation (electric shock) 3. agrobacterium ....*agrobacterium-more specific, only T DNA gets inserted into plant in agrobacterium. T DNA has left border and right border, borders tell agrobacterium where to put stuff. left and right border are repetitive sequences that bacteria read. made bacteria, now going to insert it: biolistics or agrobacterium, start with a piece of leaf tissue, hash it up so its open and exposed, insert DNA but shooting or infecting bacteria, we want this transformed into plant, totipotent-capable of giving rise to any cell type, callus tissue, using herbicide resistant to show only transformed cells are growing, add hormones so that it grows leaves, synopsis: start with leaf tissue add it to wounded leaf tissue, dedifferentiates then redifferentiates to grow into new plant
if there are many base changes to Gs and Cs, what does this mean?
it suggests adaptation to higher temperatures, because where possible the 3-hydrogen-bonding Gs and Cs have been substituted in the new genome sequences. With more G and Cs, the DNA will be more difficult to denature in high temperature environments
classify the functions or consequences of SNPs and Indels in genomic DNA
A single-nucleotide polymorphism (SNP, pronounced snip) is a DNA sequence variation occurring when a single nucleotide adenine (A), thymine (T), cytosine (C), or guanine (G]) in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. For example, two sequenced DNA fragments from different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case we say that there are two alleles: C and T. Almost all common SNPs have only two alleles. SNP diagram.png Within a population, SNPs can be assigned a minor allele frequency — the lowest allele frequency at a locus that is observed in a particular population. This is simply the lesser of the two allele frequencies for single-nucleotide polymorphisms. There are variations between human populations, so a SNP allele that is common in one geographical or ethnic group may be much rarer in another. Variations in the DNA sequences of humans can affect how humans develop diseases and respond to pathogens, chemicals, medication, vaccines, and other agents. SNPs are also thought to be key enablers in realizing the concept of personalized medicine. However, their greatest importance in biomedical research is for comparing regions of the genome between cohorts (such as matched cohorts with and without a disease). insertion-deletion: Indel is a molecular biology term for an insertion or deletion of bases in the genome of an organism. ... An indel inserts and deletes nucleotides from a sequence
What does CAR-T cell therapy treat? Describe what a CAR-T cell is and the shortcomings of the treatment
CAR-T cell therapy treats acute lymphoblastic leukemia. A CAR-T cell is a chimeric antigen receptor. CAR-T cell therapy is a type of gene therapy that involves making transgenic T-cells that have surface antigens of B cells. The patient's modified T cells are then reinserted into the patient. The T-cells recognize and kill the cancerous B cells. CAR-T cell treatment can be dangerous because it can cause cytokine release syndrome due to overwhelming T-cell activation. T cells signal using cytokines Secondly, it can cause B-cell aplasia-->where too many B cells are killed to keep the immune system functioning. Last, Tumor Lysis Syndrome may occur-->possible with ay anti-cancer treatment, abnormalities, too many cells killed at once, overwhelms system, can kill lots of things all the neighbors releasing ions and lots of things into bloodstream
example of genome editing
CRISPR
understand goal of genome editing
CRISPR -genome editing tool-changes what's already in genome (to make something not disease causing, for ex.) 2 systems 1. non homologous end joining-can often lead to changes in base pairs 2. homology directed repair-involves insertion of DNA
What enzyme is associated with CRISPR and what does it do?
Cas-9 is an endonuclease that makes double-stranded breaks to the targeted sequence
CRISPR/Cas9- how does cas9 find its target sequence, and what does it do when that sequence is located?
Cas9 protein finds its target with the help of the guide RNA sequence. The gRNA is complementary to a region of DNA. When gRNA binding occurs, the Cas9 protein will cleave the DNA, causing a double-stranded break
genome editing opinion
Genome editing techniques alter the genome of plants so that desirable traits may be inherited. Even though genome editing does not introduce biologically non-existing foreign genes to original plants like transgenic technology, it nonetheless can eliminate or replace targeted genes to select for desired traits. I am in favor of genome editing techniques because they are fast and usually accurate in creating desired results. However, I think that the FDA should monitor and document all genome editing done on plants. I do not think there should be heavy regulations on the production of edited plants, because this could slow the flourishment of disease-resistant crops. I think that loose or no regulations is acceptable for genome editing because, yes, genome editing is interfering with selection and evolution by selecting for the best traits, but it is not manipulating evolution to the extent that making new, unnatural, transgenic plants does. Genetic variation is due to mutations and recombination, and manipulating these rare and random events (in transgenic plants) can have serious consequences and influences that we cannot understand. On the other hand, I think that genome editing is much more natural, as it is using and shifting around naturally occurring genes to select for the best possible traits, as opposed to creating traits that may never have occurred without the introduction of foreign DNA. To monitor the safety of genome-edited plants, I would suggest requiring 10 or more generations of the genome-edited plant to be grown alongside a control group to ensure that only the desired trait has been effected. All DNA changes and resulting observations should be reported to the FDA to maintain a history of genome plant edits before they are mass produced. Lastly, this genome plant editing history should be kept on a public database, because this is only my opinion, and the public should be able to look up what plants have been edited, how they have been edited, and further details so that they can choose whether or not they would like to consume genome edited plants.
Explain how CRISPR + cas9 is used to edit the genome of different plant species.
Guide RNA finds matching DNA and binds to it. Cas9 cuts both strands of the DNA, resulting in a double stranded break. The DNA is then repaired using one of two repair mechanisms. The DNA can be repaired using non-homologous end join, which involves exonuclease trimming and cleaning up the DNA ends and then ligating the ends back together. This often leads to deletions, and sometimes insertions. The 2nd repair mechanism is called homologous directed repair. This involves inserting donor DNA with the same ends as the cut out DNA. the donor DNA then replaces the DNA that was cut out.
decide if you want your genome sequenced, how the typical technique works, what you can learn and what you can't
I only want to have my genome sequenced if I have an unknown disorder Genome and exome sequencing are extremely valuable in diagnosing people whose symptoms don't match known disorders. "Every time someone goes into a children's hospital with a serious disease, it would be immoral NOT to sequence him," Dr. Watson said. The first and most famous case is that of young Nicholas Volker and his intestinal condition; I've followed that of 4-year-old Gavin Stevens's blindness gene. The cases of exome sequencing solving medical mysteries are mounting fast. Steve Jobs and Christopher Hitchens had their cancer genomes sequenced, pancreatic and esophageal, respectively, and the information guided drug choices. Henry Louis Gates Jr. had his done to trace his ancestry. Glenn Close reportedly did it to better understand mental illness in her family, and I can't guess why Ozzy Osbourne did it. If someone in my family had it-to see what genes I will pass on.. but definitely do not want my entire genome sequenced ever.
Explain how Alzheimer disease in trisomy 21 individuals differs from the same disorder caused by a mutation in the APP promoter in a person who has the normal two copies of chromosome 21.
Individuals with Down syndrome typically have 3 copies of chromosome 21. that means they have 3 copies of the app gene, and develop alzheimer's as a consequence. people who have 2 copies of chromosome 21 and thus 2 copies of APP can also develop Alzheimer's, but in this case, the promoter mutation must lead to over-expression of APP. with too much APP, anyone can develop Alzheimer's, no matter how they get too much APP. down syndrome=triploid, 3 chromosome copies or one longer one, those ppl often develop alzheimers,
explain the dif between HDR and NHEJ in double-stranded DNA repair
NHEJ-non homologous end joining-involves the alignment of only one to a few complementary bases at most for the religation of two ends, easier to use, inserts or deletes mutation, changes reading so that theres a stop codon usually, gene is silenced relies on silencing pretty much??? or stops proteins HDR-homologoy Directed repair: uses longer stretches of sequence homology to repair DNA legions, much more accurate for double stranded repair, it can also introduce very specific mutations into the damaged DNA, uses DNA template in plasmid or in single stranded form, allows for accurate pair of DNA, mutation is changed to normal form and replaced =edit
List or describe 2 ways that the gene expression analysis tool of a microarray is different than a Northern blot.
Northern blots use a single probe to ask about expression of a single gene. The mRNA is immobilized in a gel, and the probe is washed over. Microarrays use 10000s of probes to ask about global gene expression; the probes are immobilized on a chip and the mRNA or cDNA is washed over the top
List or describe 2 ways that the process of PCR is the same as that of DNA replication in your cells
PCR and DNA replication both require DNA polymerase that extends the growing strand of DNA in the 5' to 3' direction. PCR and DNA both require primers to get DNA polymerase to start extension.
non-gmo
PCR based verification, targets "relevant GM events" non-profit propaganda group
use restriction enzymes and DNA ligase to clone a gene into E. coli bacteria
The process of cloning a human gene in a bacterial plasmid can be divided into five steps. 1) Isolation of vector and gene-source DNA. The source DNA comes from human tissue cells. The source of the plasmid is typically E. coli. This plasmid carries two useful genes, ampR, conferring resistance to the antibiotic ampicillin and lacZ, encoding the enzyme beta-galactosidase which catalyzes the hydrolysis of sugar. 2) Insertion of DNA into the vector. same restriction enzyme sticky ends DNA ligase.· 3) Introduction of the cloning vector into cells. Bacterial cells that are are lacZ- take up the recombinant plasmids by transformation. Some bacteria have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA. 4) Cloning of cells (and foreign genes). Plate out the transformed bacteria on a solid nutrient medium containing ampicillin and a sugar called X-gal. Only bacteria that have the ampicillin-resistance plasmid will grow. The X-gal in the medium is used to identify plasmids that carry foreign DNA. Bacteria with plasmids lacking foreign DNA stain blue when beta-galactosidase hydrolyzes X-gal. Bacteria with plasmids containing foreign DNA are white because they lack beta-galactosidase. 5) Identifying cell clones with the right gene. Sort through the bacterial colonies with foreign DNA to find those containing the gene of interest. Nucleic acid hybridization, depends on base-pairing between the gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule. Fig 20.4. A radioactive or fluorescent tag labels the probe. The probe will hydrogen-bond specifically to complementary single strands of the desired gene. After denaturation (separating) the DNA strands in the plasmid, the probe will bind with its complementary sequence, tagging colonies with the targeted gene.
USDA organic
USDA certified products indicates reduction of specific chemicals indicates absence of GMO products
define the big issue with acute lymphoblastic leukemia
White blood cells its universal, in children, crowds out blood cells, immune system -can be inherited WBCs include many immune cells that recognize "non-self" antigens B Cells, T cells, NK cells
Describe the mechanism used in BT corn that causes pesticide effects
add BT endotoxin into corn add more details ????
List possible benefits of using CRISPS-cas 9 to edit the genome of plants used to feed humans and animals
allows scientists to both remove mutations from the plants, as well as alter certain traits of the plant species
List or describe 2 ways that animal transformation and plant transformation differ
animal transformation must occur with embryos or embryonic stem cells, because these cells can differentiate into the entire organism. in plants, transformation of stem or leaf tissue can occur because all plant tissues are totipotent. animal transformation takes advantage of homologous recombination to insert genes exactly where we want them, while plant transformation gives us random insertions into the genome
example of genetic modification
artificial selection
downsides of CRISPR cas 9
may not know how plants would affect humans and animals once their genome is edited-->allergies?
compare and contrast the transformation of fertilized eggs or embryonic stem cells
biggest dif is an egg cell is transplanted directly back into the mom, give hormones to the mom. embryonic stem cells-grow outside of the body for prolonged period of time, but make a whole bunch of them outside of the mom at dif times after introduction of DNA. with the zygote or embryo that gets transformed, presume that most of the cells in that resulting baby have or not have, whereas embryonic stem cell into developing offspring the offspring will be chimera while other half might not. egg cell=100 or none. embryonic stem cells. *******inject changed DNA into egg or embryonic stem cell in animal, some percent of the time it results in transformation. put into mom, all or nothing transformation to transgenic. opposite: embryonic stem cells: now we can culture them in a dish, add an antibiotic, only the ones that live =100% transformed if they are alive, they are put inside the developing embryo, pile transformed cells in bottom of embryo-incorporated but not into whole organism, put into embryo, most babies are a transgenic-chimera, not 100% of cells will be transgenic, need to wait an entire generation for 100% of cells to be fully transgenic. most are transgenic but are chimeric. in egg/zygote 100% transgenic or 0% so its a trade off
compare and contrast biolistics and Agrobacterium-Mediatod plant transformation
biolistics-gun. agrobacterium ...both are not accurate ??????
explain the original and lab roles of CRISPR
came from bacteria, they had it to kill invading viruses, a bacterial defense mechanism, cas9 knows where to go because it has a guide RNA, then cuts the DNA apart (double stranded break), can modify gRNA-->cause "DSBs anywhere we want, can insert new DNA, cause deletions, deletions tend to be less targeted,
abstraction in synthetic biology
can order components of all different species, can think of whole unit (GFP, pig, etc.). string promoter and devices together, big picture, problem solving instead of thinking about tiny details
define CAR-T cells
carry a chimeric antigen receptor chimeric=mix of different -a form of gene therapy -involves making transgenic T cells -T-cell engineering -T cells recognize infected B cells
describe the goal of CAR-T cell therapy
carry a modified receptor that looks for B cells (leukemia).
genetically modified organisms
could be imprecise changes, something has been added, can be just through selective breeding ex: bananas modified through selection, reproduce asexually -modified by breeding, seedless watermelon-->triploid
Nickase
creates nicks in the DNA- thus requires 2 gRNAs for a double strand break, more accurate, uses NHEJ after that causes Indels, leads to insertions, deletions or frameshift
define synthetic biology
design and construct new biological parts/systems -redesign existing biological parts/systems -need strong biology background the goal-to solve problems using new models ex: tree that grows into a house
explain the process of genome editing through CRISPR/Cas9 using words or pictures
do we need to know double nickase?
how might you develop a transgenic plant where the site of integration is known?
embryonic stem cell transformation might guide you in reaching your goal
describe and diagram how homologous recombination is used to create transgenic animals
ex: microinjection into nucleus, sometimes recombination takes in new sequences. ****homologous recombination-if we have our new exciting gene, ex: a blood promoter attached to octopus ink, region A on left and region B on right, transform it into an animal that bleeds all the time, can do this in embryonic or stem cells, allows you to have recombination into chromosome, 10% of time. tragically you cannot do this in plants. same sequences recombine the same way during meiosis
modularity in synthetic biology
module (a part), parts all fit together because they are standardized
use knowledge of recombinant DNA to form an argument for or against the consumption of GMOs
for: Biotech can make food healthier, giving lettuce a greater concentration of nutrients, reducing starch in potatoes or lowering the saturated-fat content of cooking oils. Studies suggest genes introduced into GMO tomatoes can increase their natural production of antioxidants that might help prevent cancer or heart disease. Improving the nutritional values of foods can be particularly significant in boosting diets for developing countries. Through genetic modification, scientists can give crops built-in resistance to pests. That means less need for pesticides that are potentially harmful to the environment. Studies show the introduction of GMO soybean and corn in the United States led to a 13 million kilo reduction in pesticide use in the 12 years up to 2009. By reducing the need to spray, GMOs also cut farmers' fuel emissions, helping to fight global warming. against: GMOs are a serious risk to the environment. Their seeds travel well beyond fields where they are grown. Cross-pollination creates herbicide-resistant "super weeds" that threaten other crops and wild plants. Tampering with crops' genetic makeup impacts down the food chain: scientists say GMO's have decimated butterfly populations in the United States, or led to birth defects among other animals. By the time we find out the long-term impact, it could be too late.
What could explain the higher levels of TOX3 in the gel? (3)
frameshift mutation in the coding region of TOX2, mutation in the promoter of TOX3, down regulation of a repressor protein that brings to the TOX3 promoter up-regulation of an activator protein, deletion of a silencer element in the promoter, lots of other possibilities its missing a reference blot-a protein we know does not vary in cancerous tissue, so we can know that there were equal amounts of protein loaded into each of the lanes
argue for or against the use of genetic engineering and gene therapy
gene therapy is fixing something thats broken/has mutation, adding back normal gene genetic engineering is adding something new thats beneficial
define genetic engineering and genetically modified organisms (GMO)
genetic engineering-adding something new while genome editing is changing whats already there*****-focused changes, very specific ex: bananas-put a vaccine in them ex: golden rice-beta carotene ex: herbicide tolerant crops, corn soy, cotton, tobacco, rice genetic engineering is a permanent change to DNA, target embryo/fertilized egg, target embryonic stem cell genetically modified organisms (GMOs)-could be imprecise changes, something has been added, can be just through selective breeding ex: bananas modified through selection, reproduce asexually -modified by breeding, seedless watermelon-->triploid
develop a scientific position on some of the bioethical and social issues surrounding genetic engineering, especially genome editing
genetic engineering-ex: bananas w vaccines, maybe not everyone wants the vaccine, harm to nature, gets into other mammals genome editing human germline editing. This is because changes made in the germline would be passed down to future generations. The debate about genome editing is not a new one but has regained attention following the discovery that CRISPR has the potential to make such editing more accurate and even "easy" in comparison to older technologies. Bioethicists and researchers generally believe that human genome editing for reproductive purposes should not be attempted at this time, but that studies that would make gene therapy safe and effective should continue.1,2 Most stakeholders agree that it is important to have continuing public deliberation and debate to allow the public to decide whether or not germline editing should be permissible
genetic engineering
genetic engineering-focused changes, very specific ex: bananas-put a vaccine in them ex: golden rice-beta carotene ex: herbicide tolerant crops, corn soy, cotton, tobacco, rice genetic engineering is a permanent change to DNA, target embryo/fertilized egg, target embryonic stem cell
compare genome editing to gene therapy
genome editing-changing what's already in genome gene therapy-fixing something thats broken, has mutation, adding back normal gene Gene therapy is an approach whereby a new gene is transferred into cells to augment a defective gene. However, gene therapy does not remove or modify the defective DNA and is generally suited for a limited set of genetically defined diseases. Genome editing, in contrast, corrects the defective DNA in its native location. The diversity of genetic drivers of disease demands a diversity of solutions. Genome editing has the potential to deliver a variety of types of genome modification to address a broad range of genetically defined diseases. genome editing-in embryonic cell only!!!, usually heritable, which is why this has more ethical concerns genome therapy-reinsert, not heritable
example of genetic engineering
herbicide resistant crops
inserts donor DNA
homologous directed repair
describe and diagram how gene therapy is used to dress genetic mutations
infect cells with vector to change current DNA mutations
gene drive
mosquito 1 chromosome -----codes for cas9--gRNA----- chromosome 2 ----AB-----
