SynBERC:COG/Homework

What's the cloning amount (and type) per lab per week ?

Endy Lab

• Seems to come in spurts, when doing dedicated construction period we do about 8-10 assemblies / person-week. Over the course of PhD probably 1-3 assemblies / person-week. Our method is standard biobrick assembly.

Knight Lab

• Reshma 10:55, 27 November 2007 (CST): During low periods, I typically do 3-5 assemblies per week and in high periods I do 10-20 assemblies per week.

Lim Lab

• reid 17:02, 30 November 2007 (CST) When I'm cloning, I've created ~10 constructs / week (really ~40 / month in a single batch). A construct is typically 2 or 3 domains simulateneously ligated into a backbone. I use the combinatorial / type IIs restriction system that Wendell presented at the last SynBERC meeting.

What experiment can't we do that automated cloning would allow us to do?

• Jason R. Kelly 07:38, 29 October 2007 (CDT):For instance Dueber mentioned making combinatorial part libraries.

Endy Lab

• Jason R. Kelly: Making loads of characterization constructs. Would be great to be able to insert all registry promoters upstream of the same reporter and test under the same conditions. Same for RBS's, terminators, etc. If automated cloning was in place we could continue to do this as new parts came in.

Knight Lab

• Reshma 10:55, 27 November 2007 (CST): If assemblies were easier, I would probably scale up the number of combinations I try in parallel. For example, instead of rationally adjusting 1 parameter at a time (where a parameter is something like RBS strength, promoter variant etc.) ... I would try 5-10 RBS strength x 5-10 promoter variants x 3 different plasmid copy numbers x 3-5 different reporters. Easier assembly would also probably let me make better use of synthesis. i.e. I'll synthesize 10 different versions of my protein because I can easily try those versions with all the RBS, promoter, and reporter combinations. I've dabbled in this approach before but have found that the time I invest in building all these variants doesn't warrant what I get out of it (for my particular work ... it does pay off for others).

Anderson Lab

JCAnderson 15:44, 30 November 2007 (CST)Ditto to all that. I'd say in practice we do a similar volume of assembly, but we'd do far more if the tools were better.

Lim Lab

• reid 17:02, 30 November 2007 (CST) I'd echo what Dueber said about combinatorial libraries. I'd also be less conservative about characterizing a large set of previously uncharacterized promoters for a particular function (yeast mating or osmo response promoters that we don't already use in the lab, for example).

Dueber Lab

JEDueber 17:42, 3 December 2007 (CST) We do about the same biobrick throughput as above, but still frequently do multistep PCR for multipart cloning when possible. Many of our constructs are making homologous repeats so we have to avoid PCR with those.