What is the role of BSA?
Large globular protein that acts as as blocking buffer
- Preventing non-specific binding
= Reduces background noise and increases accuracy
Describe an epifluorescence microscope set up
- Light is passed through filters for a specific wavelength
- Reflected, passed through a lense and hits the object
- Is detected by a detector
Images can be stacked up to generate a 3D image
What are the technical differences between an epifluorescence
microscope and CSLM that allow the higher resolution imaging?
- Laser beam scans the specimen in CSLM to create an image, contributing to greater spatial resolution
- Light is absorbed through a very small pin hole, so out of focus light is not detected. This also reduces background noise in CSLM
What are the disadvantages of CSLM compared to epifluorescence microscopy?
- More expensive to carry out
- Takes longer to analyse a specimen
- Laser illumination could lead to photobleaching, so limit ability to capture long-term lapse images
- Due to scanning technique and time, field of view may be limited
- Less portable and bulkier than normal microscopes
Explain, briefly, how the Rubisco content has been reduced in this line.
- Antisense construct is used to target the ssu in Rubisco
- Introduces a genetic construct, complementary to mRNA
- Forming a double stranded RNA
- This is then targeted for degradation, by DICER
- Leading to a reduced expression of the small subunit of Rubisco
Describe the measurements and assumptions that underly the estimation of the CO2 concentration in the intercellular airspaces (Ci) from leaf gas exchange.
Measurements:
- Ambient carbon dioxide
- NET photosynthesis, measured as uptake of CO2
- Temperature
Assumptions:
- Steady state conditions- assumed that it reaches steady state gas exchange
- Concentration of carbon is uniformly spread through a leaf
- CO2 diffuses 1.6x slower than water
- No issue with experimental system
What is the purpose of each component in an ELISA assay?
- Sodium bicarbonate
- Ab coupled to alkaline phosphate
- Bovine serum albumin (BSA)
- Positive control solution
- Sodium bicarbonate
= aids solubility and stability of proteins, acts as a buffer - Ab coupled to alkaline phosphate
= Ab for specific recognition, enzyme catalyses pNPP to a yellow product - Bovine serum albumin (BSA)
= Blocks non-specific binding - Positive control solution
= Checks assay works
What are the advantages of analysing CO2 assimilation as a function of Ci rather than CO2 surrounding the leaf?
- Stomatal conductance taken into account
- May vary in response to the environment
- More accurate
What are the role of the following in DNA extraction?
- Lithium chloride
- w/v SDS
- isopropanol
- 70% ethanol
- Lithium chloride
= salt to neutralise the charge of the DNA, more likely that DNA will separate and form a pellet - w/v SDS
= Detergent, breaks down membranes and releases DNA from nucleus - isopropanol
= Precipitates DNA - 70% ethanol
= Remove the salt from earlier as this would affect PCR
Why should the student include a protein ladder/standard when running the gel?
- To use as an indicator/calibrator for protein size
- Can be used to determine the molecular weight of proteins
Why should samples be kept on ice?
- Prevention of enzymatic activity - slowing down reactions
- Minimize protein degradation
- Reduction in protease activity
- Prevent sample oxidation and oxidative damage
- Helps preserve native protein structure
What could they be doing incorrectly when pipetting to not get the right value? Provide a suggestion for how they could improve their technique
- in initial uptake be pushing down hard to the stop and then drawing up liquid
(because all the values are greater)
A disc removed from the leaf is imperfect. What should the demonstrator advise?
- Try again and get a new disc
- As it may effect accuracy and amount of DNA/ protein content extracted
What are the key components of a figure legend?
Title
Techniques (PCR/Gel/ etc.)
Groups/ Treatments (WT, mut)
Result (line, increase, significant difference)
Other details on experiment (e.g. Temp)
Explain briefly how mechanical homogenisation and incubation temperature can impact recovery of sucrose
Mechanical homogenisation
- physical breakdown of cell wall and membrane to release sucrose
- increase recovery
Incubation temperature
- more extreme temperatures will denature the membrane
- higher will lead to more movement and gaps, increasing sucrose recovery
- snap-freezing, can render the wall and membrane brittle, also increasing recovery
To understand the function of CYCLOPS, it is necessary to generate loss-of-function mutants via CRISPR-Cas9. Describe the main steps and key considerations in the process of generating homozygous cyclops mutants in rice
CRISPR/ Cas9
1. Generate a guide RNA, which will guide it to the CYCLOPS gene
2. Transform the rice
- Achieved by A.tumefaciens
3. Select for mutants (positive transformants, by applying a selection pressure
4. Cross mutant heterozygous plants
5. Check by screening for homozygous e.g. PCR or NGS
RNAi
- introduce an RNA transcript that will post-transcriptionally silence the CYCLOPS gene
- Can be self crossed to generate homozygous mutants
Why does determination of maximal photosystem II (PSII) efficiency require dark-adaptation?
- All the PSII reactions centres are open, ready to accept electrons
- Represents baseline/ ground state (F0)
- Minimal NPQ, due to the lack of light
- Steady state because it has been in darkness for enough time to be uniformly adapted
How is it possible that photosystem II subunit S (PsbS) overexpression mutants have much higher NPQ values than wild-type plants, yet very similar PSII operating efficiency?
- The role of PsbS is to activate NPQ by dissipating excess energy
- Overexpression generates a greater capacity to initiate NPQ
= More rapid response to high light is signalled - So the NPQ values are higher
- Operating PSII is a signal of plant health, and overexpression doesn’t effect this
- PsbS is only involved in photoprotection when levels of light are too high, however doesn’t effect the normal function of PSII
What is the role of ICS1 and ICS2 gene?
Isochorismate synthase 1 & 2
- Isoforms of each other
- Important in the biosynthesis of salicylic acid (SA)
- Exhibit a degree of functional redundancy
What are the requirements for a primer?
- 3’ CG clamp
- 20bp minimum length
- Equal distribution of A,T,C,G
- No palindromes (same backwards + forwards)
- No homopolymeric sequences
- No self complementary regions
- Tm of 50-60°C
Why might an adult leaf, have a greater sucrose content than younger leaves?
Storage location
Mature- enhanced chloroplast structure
- Higher expression of enzymes
What gene fusions will you use to measure the timing of PNE1 expression over a vircadian time course in wild type and blackpool null you use? How would you visualise this in the plant?
- To measure over a time course, fuse the PNE1 promoter to a luciferase gene
- Measure luminescence with a luminometer
- Luminescence is only generated when the promoter is being activated
Describe the main differences in the transformation methods used for Arabidopsis thaliana and Nicotiana tabacum (tobacco).
- Both can be transformed using A.tumefaciens, which involves the insertion of a plasmid
- Different promoters and selection markers
- Biolistics may be used instead for plants that are recalcitrant to A.tumefacien transformation
Why would an adult leaf have less sucrose than a young leaf?
- Lower metabolic activity
- Sink- source dynamic, sucrose is moved away
- More used for storage of starch instead of shorter term energy (sucrose)
