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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 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.

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

4. Making new parts by PCR

C. French

• 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.

• 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.

• 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

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.
C. French

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
• 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.

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

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.
C. French

• 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.

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.

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Outsource your assembly

• Gingko Bioworks

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