9th July 2009
Calcium encapsulation; in yeast cells (S.cerevisiae); use artificial cell. Create artificial cell for yeast, control time that it releases the molecules for auto-encapsulation. Individual cell coated with calcium layer. Layer by layer process.
Cells enter stationary phase and extends their lifetime. Use as scaffold.
Embed into calcium carbonate layer.
Design membrane protein covered in nucleation sites and control release.
Drugs (oral delivery - dissolve coat in stomach acid, deliver drug. Ideally cell would be dead, but required drug contents would be present and released upon breakdown on coat), storage, improve efficiency of transport. Burn off with acid. Put acidic receptors - can model and adjust parameters.
Paper looked at artificial way. Charge to outer layer for deposition at nucleation sites. Adapt this to enable the cell to do it itself.
- Look more into coat formation.
- Find any existing membrane proteins that could be upregulated and singly negatively charged amino acids.
- Release, transport, application.
- Probiotic release of myelrosinase?
- pH at which shell is broken down and induction of restriction enzyme to break down all DNA in the chassis.
- Once encapsulated is there possible reaction with anything?
- Drug synthesis?
Highly mutatable polymerase.
Use GFP protein to select. Strength of fluorescence determines whether mutation has increased or decreased fitness.
Intuitively pick best function.
Parallels with theories in systems biology. Introduce mutations to optimise the circuit. Link in with idea of small world networks - optimisation of systems. Test robustness of genetic circuits.
- Determine nodes/genetic circuit to be optimised.
- Jumping genes/transposons.
- Find more rational approach - determine exactly how to optimise system rather than using directed evolution.
- As a stress test?
Hydrogen from Biomass
magA...! Full two-page report from James. Increase hydrogen yield.
- How is it SB more than metabolic engineering.
- Other solutions to producing hydrogen - diatoms. Used currently in water purification - production of hydroxy radicals.
- Difficulty in separating titanium oxide from water - use magnetite and use this to remove from water. Reduces efficiency.
- Added layer of silicon to overcome this - retain magnetic properties but doesn't heat up. Could introduce magA to diatoms?
- Genetic engineering approaches to produce hydrogen.
Harvard '08 iGEM project.
Produce electricity. Wild-type - mtrB gene. Wild type which contains the mtrB gene produces more electricity. Increase temperature and more electricity is produced.
Also, microbial fuel cell.
Exact mechanism unknown. But knock out certain genes or introduce others can increase electricity production.
Anaerobic anode, exploitation of lac to transfer electrons to cathode.
Different classes of bacteria that produce electricity.
- Find applications.
- Difficulties surrounding chassis and which strain would be used.
- Combination with magnets?
- Find novel and exciting application.
- Stabilisation of production?
- Electricity as output module of camera idea or magnetometer.
- Use as quantification tool?
Each bacteria only produces a small amount of electricity. This is too little to measure.
1) Macroscale measurement of many cells
2) NMR type method to take electricity measurements from all cells, but discern the amount produced from each cell
so each electric signal is like a proton signal in a spectrum, which can be further analysed and attributed
3) Couple output to a flagellar rotor and measure torque/speed relation. Click on link for more!
Couple with output to computer.
Level of fluorescence.
Heigh resolution of cell input and output.
Measure high numbers of cells, eg each cell acts as a pixel.
- Think of what to detect.
- How precise would it be?
- How to get as high a resolution as possible?
- Possibility of measurement of pH of single cells. Although pH will diffuse.
- Have source of electrons as output.
- Link to flagella?
- Why link biology to electronics? Reasoning - Why have a biological camera?
Problem to be solved
When genes are heterologously expressed, the expression levels are lower than optimal.
cells produce a protein on interest together with a signal molecule.
The promoter and protein gene will undergo limited mutation by T7 DNA polymerase, so that the expression levels of the protein will change.
The signal molecule will include information about how much protein is being produced.
(the amount of signal molecule will need to correlate with amount of protein, and will also need to take into account the diffusional reduction in concentration)
The cell with the highest amount of protein production will know, and it will stop mutation. (critical point)
all the other cells will however carry on mutating. This ensures that the most highly optimised cell (to that point) will not mutate further and result in lowering of protein production.
This process of mutation iterates until one cell remains with the highest amount of protein production for an extended period of time
1) random mutation generates many non-functional/different proteins.
thus need a selection mechanism for the correctly expressed proteins
2) functionality is more important that increases in yield
3) self-optimisation can be completed more easily by current companies that use genetic algorithms
to maximise codon usage and RBS stability
Using directed evolution to increase tolerance of a cell (e.g. yeast in fermentation) to ethanol. Selected evolution of a specific gene using hAID and polymerase.
hAID is used in antibody production to create hypermutation in the receptor of the antibodies. From this method antibodies to a wide range of antigens can be created. We aim to use hAID combined with specific polymerases to target those genes associated with proteins for cell viability (especially those for ethanol resistance). Through hypermutation of these genes, and using an evolutionary selection pressure of increasing ethanol concentration. In this way, cells with ineffective protein defense mechanisms will be killed, whereas those with increased resistance will survive and proliferate. This would hopefully increase the tolerance to ethanol from the current strains (vary from 5%-21% by strain of yeast). Research is needed into the areas of the cell that ethanol targets. Are there any specific ethanol resistance proteins that can be targetted?
Could also be used for increasing tolerance to different disinfectants/ antibiotics (although increasing antibiotic resistance clearly NOT ethically sound!)
Further research shows that ethanol targets many different areas within the cell, and many remain partly unclear. For this reason it may not be feasible to engineer a single resistance protein to increase tolerance. We also felt that this 'brute force' evolution was not a very elegant solution to the problem, and not likely to succeed.
Important points to take from this research are the possibilities for directed evolution of specific genes. This may well come in useful for other projects.
WORK IN PROGRESS
Twittering is a micro-blogging website were people can post short (<140 characters) updates about themselves and their activities that are then read by "followers" who follow your updates. For those of you who know facebook, twitter is comparable to the status bar in a facebook profile.
The project aims to create an interface between the biological and the computational worlds through the use of modulable reporters engineered in an E. Coli chassis.
A novel toolbox populated by both Biobricks dictating the modularity of detection by the computer as well as software programs that do the processing steps.
Genes of Interest
- Photosynthetic slugs
Very useful resources
I just went to a talk where they briefly mentioned this database. We can find proteins that many genes code for: Gene ontology
Also, "ecoli wiki": Ecoli wiki