Mix up genomic library with cell-free expression system and oil.
End up with oil droplets with DNA plus expression system.
Advantages? Not sure
Apparently better than other in vitro selection techniques such as phage display/ribosome display/mRNA peptide fusion/SELEX (would like to read about these too)
----
Paper on DNA shuffling of Fe-Fe hydrogenases
-> heterologous expression of h2ase in E coli:D
Looks to be similarly possible for Ni-Fe.
---
Directed evolution of oxygenases:
Table 1 compares selection methods
FACS pretty good (10^7). Phage display probs wouldn't work cos the protein wouldn't fold properly. Solid media a bit iffy, but I think it would work well if there was a selection pressure on the cells.
----
Monday, February 22, 2010
Tuesday, February 16, 2010
Enzyme (re)design: lessons from natural evolution and
computation
John A Gerlt1 and Patricia C Babbitt
"Aggressive enzyme engineering". Instead of making small changes, tear off massive chunks of the protein and add on new loops.
Use computers (RosettaMatch) to work out what loops will work well on what scaffold.
Follow it with directed evolution to optimise function.
computation
John A Gerlt1 and Patricia C Babbitt
"Aggressive enzyme engineering". Instead of making small changes, tear off massive chunks of the protein and add on new loops.
Use computers (RosettaMatch) to work out what loops will work well on what scaffold.
Follow it with directed evolution to optimise function.
Wednesday, February 10, 2010
"Enzyme (re)design: lessons from natural evolution and
computation"
- functionally diverse superfamilies: same catalytic method, different reactions
- can create new catalysts by taking an original enzyme and modifying its specificity (same catalytic method)
- usually occurs at expense of original functionality
- other option: some enzymes are promiscuous: use directed evolution to favour one reaction.
computation"
- functionally diverse superfamilies: same catalytic method, different reactions
- can create new catalysts by taking an original enzyme and modifying its specificity (same catalytic method)
- usually occurs at expense of original functionality
- other option: some enzymes are promiscuous: use directed evolution to favour one reaction.
Thursday, January 21, 2010
Wednesday, January 20, 2010
9000 turnovers per second = from Desulfovibrio vulgaris.
Glucose transporter: "
http://www.kazusa.or.jp/codon/ - "Count Codon" service
Inosine = universal base pair
Stratagene Mutazyme = good system for ePCR (less mutational bias - inherently error prone polymerase)
Functional proteins from a random-sequence library: completely random sequence of DNA, combined with mRNA display (?) allowed the generation of a novel ATP binding protein:O
Directed Evolution of Novel Protein Functions:
- From a theoretical perspective, the frequency of functional proteins in the entire protein sequence space is high enough that novel proteins could be generated simply by random methods (but still, low frequency). Pretty cool! If you can come up with a screening mechanism, then you can generate proteins that can do anything:O
- Recognising that it may take several sequential mutations to achieve a desired function, Chen and Zhao developed a screening methodology that involves iterative experiments that attempt to develop a function protein through a series of logical, intermediate steps
- To develop novel proteins, only a small number of mutations are necessary and these are normally localised to the active site
"GC clamp" - add a GC rich region next to sequence of interest. Use in primers to enhance binding to sequence (GC bonds = stronger)
Directed evolution of biocatalytic processes:
- Improvements in one trait may be offset by decline in another trait
- In order to generate useful industrial biocatalysts, screening conditions in directed evolution experiments should closely mimic those that will be found in the industrial setting. Can perform microscale experiments that can be scaled up to large bioreactors
- It's quite hard to ligate mutagenised DNA fragments into a plasmid to transform an E coli - ie you lost a fair bit of your mutation realm simply due to the inefficiency of ligation and transformation
Directed evolution of industrial enzymes
- 'you get what you screen for'
Glucose transporter: "
Trophic Conversion of an Obligate Photoautotrophic Organism Through Metabolic Engineering"
http://www.kazusa.or.jp/codon/ - "Count Codon" service
Inosine = universal base pair
Stratagene Mutazyme = good system for ePCR (less mutational bias - inherently error prone polymerase)
Functional proteins from a random-sequence library: completely random sequence of DNA, combined with mRNA display (?) allowed the generation of a novel ATP binding protein:O
Directed Evolution of Novel Protein Functions:
- From a theoretical perspective, the frequency of functional proteins in the entire protein sequence space is high enough that novel proteins could be generated simply by random methods (but still, low frequency). Pretty cool! If you can come up with a screening mechanism, then you can generate proteins that can do anything:O
- Recognising that it may take several sequential mutations to achieve a desired function, Chen and Zhao developed a screening methodology that involves iterative experiments that attempt to develop a function protein through a series of logical, intermediate steps
- To develop novel proteins, only a small number of mutations are necessary and these are normally localised to the active site
"GC clamp" - add a GC rich region next to sequence of interest. Use in primers to enhance binding to sequence (GC bonds = stronger)
Directed evolution of biocatalytic processes:
- Improvements in one trait may be offset by decline in another trait
- In order to generate useful industrial biocatalysts, screening conditions in directed evolution experiments should closely mimic those that will be found in the industrial setting. Can perform microscale experiments that can be scaled up to large bioreactors
- It's quite hard to ligate mutagenised DNA fragments into a plasmid to transform an E coli - ie you lost a fair bit of your mutation realm simply due to the inefficiency of ligation and transformation
Directed evolution of industrial enzymes
- 'you get what you screen for'
Monday, January 18, 2010
Ultrahigh-Throughput FACS-Based Screening for Directed Enzyme Evolution
Wiley InterScience :: Article Full Text HTML: "Ultrahigh-Throughput FACS-Based Screening for Directed Enzyme Evolution"
- use GFP as reporter gene, then run through FACS machine.
SCOPE:
Shuffle non-homologous proteins
- use GFP as reporter gene, then run through FACS machine.
SCOPE:
Shuffle non-homologous proteins
Subscribe to:
Comments (Atom)