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C. French
BioBrickMasterClass21Jul10.dociGEM UK Meeting21 July 2010Making and using BioBricks
1. iGEM is a
community development activity
. As part of your project you
MUST
:
•
make some BioBricks which will be useful to the community.
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use RFC10 assembly standard (or obtain a variance).
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deposit your BioBrick DNA in the Repository.
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characterize the operation of your BioBricks to help future users.
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deposit the characterization data in the Registry (not just on your team wiki!).
2. Getting parts from the Registry
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Use the search function to find the part you want.
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Check the characterization data.
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Go to ‘Get this BioBrick’.
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If the BioBrick is in the Spring 2009 distribution, the well will be indicated.
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Check the sequence to make sure that the part is what it is should be.
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Use DNA to transform highly competent cells. Methods on OpenWetware.Make sure you use the right antibiotic.
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If not, you can request the BioBrick from hq@igem.org. This may involve
some delay.
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Parts from iGEM 2009 do not seem to be in the distribution – might be fastestto write to the team and request them.http://partsregistry.org/Main_Page
3. Getting parts synthesised (2009 information)
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GeneArt is sponsoring iGEM
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Each team can get a certain amount of DNA synthesised for half price (lastyear this worked out to E0.20/bp).
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Maximum size of each fragment 1 kb
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Set up account at ‘Mr. Gene’.
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Allow plenty of time! Average turnaround is stated as 15 days, but someteams have had bad experiences.
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Avoid repetitive sequence.
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Will need to transfer to standard Registry vector.
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Small BioBrick (promoters, RBS) can be synthesised as two complementaryoligonucleotides.http://2009.igem.org/Partner_Offers
4. Making new parts by PCR
Template
C. French
•
is the organism available? DSMZ is cheaper than NCIMB which is muchcheaper and faster than ATCC.
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will you be able to grow it?
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can you just obtain the DNA from another researcher?
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cell suspensions usually work better than purified gDNA.
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Genomes with high GC may require special conditions.http://www.dsmz.de/http://www.ncimb.com/http://www.lgcstandards-atcc.org/Primers
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make sure you get the prefix and suffix right
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assembly standard 10 is recommended, or 23 if fusion proteins are needed.
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if you want to use a different assembly standard, you must obtain a variancefrom iGEM HQ. Apply as early as possible.http://partsregistry.org/Help:Contentshttp://partsregistry.org/Help:BioBrick_Prefix_and_Suffixhttp://www.biobricks.org/Polymerase
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Taq is fast and cheap and leaves A-overhangs, but has a strong tendency tointroduce mutations.
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Pfu is much more accurate but slower and more expensive.
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New polymerase such as Kod, Velocity and Phusion are fast and accurate.Reaction conditions
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Templates with strong secondary structure can be amplified by increasing thedenaturation time to 1 minute per cycle and including 10% v/v glycerol in thereaction.Cloning your PCR products in a plasmid vector
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Direct digestion of ends and insertion into Registry vector.
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TA-cloning: only works with Taq or some blends.
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TOPO-cloning: works for blunt-ended products.
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Clontech InFusion (ligase-independent cloning)
5. Site-directed mutagenesis
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Internal EcoRI, XbaI, SpeI and PstI sites must be removed.
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NotI sites: status is unclear.
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Strategene Quickchange kit.
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MABEL: MutAgenesis with Blunt-Ended Ligation is fast, easy, cheap andreliable (so far). See my OpenWetware site for the protocol.
C. French
6. Sequence checking
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Sequence early, sequence often!
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Confirm identity of clone: just because it is the right size does not mean it isthe right DNA!
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Check for PCR-induced mutations or sequence variations.
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Result of sequencing can be deposited in the Registry with the BioBrick.
7. Registry Vectors
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All BioBricks must be submitted in standard Registry vectors.
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Registry vectors are listed as ‘plasmid backbones’
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eg pSB1A3: Set 1, A=ampicillin resistance, version 3.
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also available in chloramphenicol, kanamycin and tetracycline resistanceversions.
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all vectors are available with a
ccdB
cell death gene (requires propagation in aspecial host strain) for efficient insert exchange.
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also available with RFP gene for red-white selection.
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Rumor has it that only parts in pSB1C3 will be accepted in 2010, but there iscurrently no text in the DNA submission page.http://partsregistry.org/Plasmid_backboneshttp://partsregistry.org/Plasmid_backbones/Assembly
8. Assembling two parts
Important noteLigations give you a complicated mixture of products. The problem in assembly is toselect the right one. This can be done either by cloning or PCR.Standard method
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excise the upstream part with E/S, cut the downstream construct with E/X, andligate.
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Or, excise the downstream part with X/P, cut the upstream part with S/P, andligate.
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Problems: often low efficiency, no way to select: many minipreps!http://partsregistry.org/Assembly:Standard_assemblyThree-antibiotic method
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Excise upstream part with E/S, excise downstream part with X/P, and ligateinto a vector cut with E/P which carries a different antibiotic resistance marker to those of both the upstream and downstream parts.
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Perform triple ligation.
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May require vector exchange.
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