Assignment Task:
Q1 (10 points) Your aim is to derive a homozygous seed stock of plants expressing transgene X ONLY (=> you have to find a way how to eliminate plant selectable marker).
You transformed canola plants using particle bombardment as the method of transformation. Canola, a diploid species, is an obligatory cross-pollinator (cannot self-pollinate). Draw transformation vector(s) (labelling transgenes as PSM and Gene X will be sufficient) and design a breeding protocol that will result in XX homozygous plants starting from a T0 primary transformant that carries a single insertion of each transgene.
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Write My Essay For Me- Identify parental genotypes that are used to establish a segregating population (population that segregates PSM and gene X) and explain how parental lines have been derived
- Show plant genotypes of relevant loci at every stage of breeding
- State the way you will identify genotypes in segregating population
Q2 (15 points) Enzymes A and B have a specific antibacterial effect on bacteria X and, thus, can be used as drugs against this pathogen. Enzyme B has identical sequence as enzyme A (domain 1) but has a 50-amino acid extension at carboxyl terminus of the protein (domain 2, see schematic bellow).
Domain 1 Domain 2
Enzyme A
Enzyme B
The junction between domains 1 and 2 in Enzyme B has been shown to be flexible, and addition of up to 10 amino acids does not abolish the function of enzyme B.
There are two strains of bacteria X (X1 and X2); enzyme A is specifically effective only to strain X1, and enzyme B to strain X2. The presence of bacterial strains X1 and X2 are unpredictable.
You want to create transgenic tomato that can be used as edible drug! You want to be able to grow your crop and, prior to flowering, decide if plant should produce fruits expressing enzyme A or enzyme B, so that you can produce drug matching the bacterial strain that is currently present.
- Draw constructs, including all transgenes, required for creation of such transgenic tomato and provide a short description of your strategy;
b) Provide a schematic and a short description of how you’ll produce tomato fruits expressing enzyme A or enzyme B if bacterial strains X1 or X2 are present.
Q3 (10 points) Salmonella is a common household bacterium that causes the food borne illness salmonellosis. Salmonellosis results in many costly and unnecessary emergency room visits each year. You have discovered that the VirA gene in one of the Agrobacterium strains has undergone rearrangement such that extracellular domain (which normally binds the plant cell wall component) has been exchanged with a domain that binds a protein specifically found on the cell surface of Salmonella bacteria. You refer to that modified VirA gene as VirA-Sal. Existence of this novel VirA-Sal gene/protein has given you the idea that this Agrobacterium mutant can be used as a Salmonella biodetection kit. The idea is that you will create a plate covered with Agrobacterium cells that express VirA-Sal. When this plate comes in contact with a food sample containing Salmonella, you would like the Agrobacterium cells to fluoresce green, signaling the presence of Salmonella.
Can you build a Salmonella biodetection kit? Draw a transgene cassette and give description of approach.
Q4 (10 points) The weed XY is one of the world’s major problems in agriculture. Resistance cases to most herbicide classes have been reported for this plant. Management of this weed in agroecosystems relies on a better understanding of how it copes with herbicide toxicity. You want to use the CRISPR-Cas9-genome editing to knock out a cluster of nine P450 genes shown on schematic below……. The weed is transformable by Agrobacterium.
DNA sequence A (only one strand is shown):
ATGGGAAGAA AAAAACTAGA AATCAAGCGA ATTGAGAACA AAAGTAGCCG
ACAAGTCACC TTCTCCAAAC GTCGCAACGG TCTCATCGAG AAAGCTCGTC
AGCTTTCTGT TCTCTGTGAC GCATCCGTCG CTCTTCTCGT CGTCTCCGCC
TCCGGCAAGC TCTACAGCTT CTCCTCCGGC GATAACCTGG TCAAGATCCT
DNA sequence B (only one strand is shown):
TGTTCAACTG GAGGAACACC TTGAGACTGC CCTCTCCGTG ACTAGAGCCA
AGAAGACCGA ACTCATGTTG AAGCTTGTTG AGAATCTTAA AGAAAAGGAG
AAAATGCTGA AAGAAGAGAA CCAGGTTTTG GCTAGCCAGA TGGAGAATAA
TCATCATGTG GGAGCAGAAG CTGAGATGGA GATGTCACCT GCTGGACAAA
- Can you design the target-specific crRNA sequence (red sequence in the schematic below) for the sgRNA you will need for your experiment? Can you briefly explain how did you derive the sequence?
- Draw and label elements within the transgene cassette that you want to use to delete a cluster of nine P450 genes.
- How would you screen for weed transformants?
- (OPTIONAL: How would you screen for weed plants in which the cluster of nine P450 genes has been deleted?)
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