Notes on Tom Knight's talk
(it would be nice to podcast our talks)
- Books that Tom Knight recommends for folks getting started
- Mark Ptashne's -- A Genetic Switch (get's down and dirty about the mechanisms, but also begins to talk about abstraction). Get the third edition.
- Benno Muller-Hill's book -- The Lac Operon.
- Fred Neidhardt's book on bacterial physiology. Physiology of the Bacterial Cell.
- Kathy Barker's book calldc "'At The Bench"' from Cold Spring Harbor Bench is a great book for naive folks walking into the lab.
- Methods for General and Molecular Bacteriology -- great book on culturing bacteria and keeping them alive. Aimed more at the industrial applications, but also a lot about methodology.
- Tom's history/experience as he set out to start engineering biology.
- General Introduction --- initial tremendous frustation in biology. every experiment turned into two experiments. first, there was the experiment that you wanted to do. second, there was the experiment that you had to do in order to do the experiment that you wanted to do? e.g., will this restricition enzyme work with this DNA, or, is it not methylated, or something else, or something else. Basically, there are too many things to worry about. think about LEGOS. everything is designed to go together. even the flowers snap together.
- Standard Assembly -- Here's the link to the original article...
Here's the link to the Registry webpage about standard assembly...
Says that this is one arbitrary thing to do, that there are many other possibilities. The relevance of this is that somebody (i.e., Tom) has already thought through the issues that you need to deal with in order to get your cloning to always work.
- BioBricks have standard cloning sites that bracket each part. The sites are layed out as EcoRI, XbaI <PART> SpeI, PstI.
- The feature of these restriction sites is that you can take two parts A and B and make an AB or BA chimera and, having done that, you are left with a new part that has the same four bracketing restriction sites. This means that you can keep adding parts to parts and so on. This is called idempotent assembly.
- TK mentions that the restriction sites are also selected to be insensitive to bacterial methylases. For example, at the right end of the XbaI site, TCTAGA, if left blank at the end, could produce a situation where a part began with TC, thus creating a GATC methylation site. Thus, the TCTAGA site is bracketed by a standard extra base (G in this case) such that it is impossible to make a GATC...
- Talks about requirement that, by defition, parts must not encode any of the standard restriction sites. (this hasn't yet ever been a problem for an open-reading frame, but in at least one case -- M0100, a tRNA reporter -- has limited the full functioning of the part in the assembly process (i.e., it could only be prefixed or suffixed).
- Talks about barcodes. Barcodes are extra bits of DNA that accompany open reading frames. They might be useful for detecting fragments of DNA, testing to see if they have BioBricks in them or not -- Here's the link to the Registry Page about barcodes
- Talks about plasmid naming scheme. pSB#N#, where the first # refers to the replication origin, the letter N refers the antibiotic resistance, and the second # refers to the cloning site. Here's the link to a summary of the BioBricks plasmid naming scheme
- Talks about the ccdB insert that is sometimes in the standard vectors. When a plasmid has this insert it's labeled as pSB#N#-1 (i.e., the extra "dash one"). So, for example, pSB2K3-1.