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C.

French

BioBrickMasterClass21Jul10.doc
iGEM UK Meeting
21 July 2010

Making 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.
• use RFC10 assembly standard (or obtain a variance).
• deposit your BioBrick DNA in the Repository.
• characterize the operation of your BioBricks to help future users.
• deposit the characterization data in the Registry (not just on your team wiki!).

2. Getting parts from the Registry

• Use the search function to find the part you want.


• Check the characterization data.
• Go to ‘Get this BioBrick’.
• If the BioBrick is in the Spring 2009 distribution, the well will be indicated.
• Check the sequence to make sure that the part is what it is should be.
• Use DNA to transform highly competent cells. Methods on OpenWetware.
Make sure you use the right antibiotic.
• If not, you can request the BioBrick from hq@igem.org. This may involve
some delay.
• Parts from iGEM 2009 do not seem to be in the distribution – might be fastest
to write to the team and request them.

http://partsregistry.org/Main_Page

3. Getting parts synthesised (2009 information)

• GeneArt is sponsoring iGEM


• Each team can get a certain amount of DNA synthesised for half price (last
year this worked out to E0.20/bp).
• Maximum size of each fragment 1 kb
• Set up account at ‘Mr. Gene’.
• Allow plenty of time! Average turnaround is stated as 15 days, but some
teams have had bad experiences.
• Avoid repetitive sequence.
• Will need to transfer to standard Registry vector.
• Small BioBrick (promoters, RBS) can be synthesised as two complementary
oligonucleotides.

http://2009.igem.org/Partner_Offers

4. Making new parts by PCR

Template
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• is the organism available? DSMZ is cheaper than NCIMB which is much


cheaper and faster than ATCC.
• will you be able to grow it?
• can you just obtain the DNA from another researcher?
• cell suspensions usually work better than purified gDNA.
• Genomes with high GC may require special conditions.

http://www.dsmz.de/
http://www.ncimb.com/
http://www.lgcstandards-atcc.org/

Primers
• make sure you get the prefix and suffix right
• assembly standard 10 is recommended, or 23 if fusion proteins are needed.
• if you want to use a different assembly standard, you must obtain a variance
from iGEM HQ. Apply as early as possible.

http://partsregistry.org/Help:Contents
http://partsregistry.org/Help:BioBrick_Prefix_and_Suffix
http://www.biobricks.org/

Polymerase
• Taq is fast and cheap and leaves A-overhangs, but has a strong tendency to
introduce mutations.
• Pfu is much more accurate but slower and more expensive.
• New polymerase such as Kod, Velocity and Phusion are fast and accurate.

Reaction conditions
• Templates with strong secondary structure can be amplified by increasing the
denaturation time to 1 minute per cycle and including 10% v/v glycerol in the
reaction.

Cloning your PCR products in a plasmid vector


• Direct digestion of ends and insertion into Registry vector.
• TA-cloning: only works with Taq or some blends.
• TOPO-cloning: works for blunt-ended products.
• Clontech InFusion (ligase-independent cloning)

5. Site-directed mutagenesis

• Internal EcoRI, XbaI, SpeI and PstI sites must be removed.


• NotI sites: status is unclear.
• Strategene Quickchange kit.
• MABEL: MutAgenesis with Blunt-Ended Ligation is fast, easy, cheap and
reliable (so far). See my OpenWetware site for the protocol.
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http://www.openwetware.org/wiki/French_Lab

6. Sequence checking

• Sequence early, sequence often!


• Confirm identity of clone: just because it is the right size does not mean it is
the right DNA!
• Check for PCR-induced mutations or sequence variations.
• Result of sequencing can be deposited in the Registry with the BioBrick.

7. Registry Vectors

• All BioBricks must be submitted in standard Registry vectors.


• Registry vectors are listed as ‘plasmid backbones’
• eg pSB1A3: Set 1, A=ampicillin resistance, version 3.
• also available in chloramphenicol, kanamycin and tetracycline resistance
versions.
• all vectors are available with a ccdB cell death gene (requires propagation in a
special host strain) for efficient insert exchange.
• also available with RFP gene for red-white selection.
• Rumor has it that only parts in pSB1C3 will be accepted in 2010, but there is
currently no text in the DNA submission page.

http://partsregistry.org/Plasmid_backbones
http://partsregistry.org/Plasmid_backbones/Assembly

8. Assembling two parts

Important note
Ligations give you a complicated mixture of products. The problem in assembly is to
select the right one. This can be done either by cloning or PCR.

Standard method
• excise the upstream part with E/S, cut the downstream construct with E/X, and
ligate.
• Or, excise the downstream part with X/P, cut the upstream part with S/P, and
ligate.
• Problems: often low efficiency, no way to select: many minipreps!

http://partsregistry.org/Assembly:Standard_assembly

Three-antibiotic method
• Excise upstream part with E/S, excise downstream part with X/P, and ligate
into a vector cut with E/P which carries a different antibiotic resistance marker
to those of both the upstream and downstream parts.
• Perform triple ligation.
• May require vector exchange.
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http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly

BALTIC
• An assembly method using an extra restriction site in the vector outside the
BioBrick region, usually ScaI.
• High efficiency.
• Requires ScaI sites to be absent from both parts.
• See my OpenWetware site for details.
• superceded now, but might still be interesting if you are having problems with
a particular construct.

http://www.openwetware.org/wiki/French_Lab

RIVAL: in vitro assembly


• Digest upstream part with SpeI, downstream with XbaI, and ligate.
• Use ligation as template for PCR with forward primer from upstream part,
reverse primer from downstream part.
• Purify PCR product and EITHER clone as if it were a new BioBrick, OR add
it to another part in the same way.
• Can add several parts in rapid succession (1 or 2 per day), including a vector,
before cloning final construct.
• Need primers for all parts, even pre-existing ones.
• Danger of PCR-induced mutations.


uuuuuuuuuuuuuuuuuuuu-a ctaga-ddddddddddddddddd
uuuuuuuuuuuuuuuuuuuu-tgatc t-ddddddddddddddddd

Outsource your assembly


• Gingko Bioworks

http://ginkgobioworks.com/

9. Characterizing your BioBricks

• Tests for function.


• eg Jason Kelly’s promoter characterization kit: compares promoter activity to
a standard promoter under standard conditions.
• You must characterize at least one BioBrick! Promoters and enzymes are
easiest in most cases.

10. Documenting your BioBricks

• Enter characterization data IN THE REGISTRY, not just on your wiki!


• Add links from your wiki to the Registry, rather than the other way around.
• At minimum, set the flag for ‘Works’.
C. French

Here Endeth The Lesson