Talk:Synthetic Biology:Vectors/Single copy plasmid

Location of primer binding sites

I was wondering about the arrangement of the VF/VR sites and the terminators. If the terminators were outside the VF/VR sites we would get somewhat longer sequencing reads. The way they are designed currently I suppose there would be less possibility for non-specific transcription so there are arguments either way --BC

• Currently the VF2 and VR sites lie outside the terminators because typically sequence data take at least ~100bp to become clear and easy to read. Therefore the space between the primer binding sites and the part is intentional and we may as well put the terminators there as anything else. It could be that it is possible to move the primer binding sites closer to the part without sacrificing read quality. I'll need to check old sequence data to see how much "extraneous" (i.e. vector) sequence appears in my sequence reads.-- RS
• In examining old sequence data, it appears that it takes at most 35 bp between the end of the sequencing primer and the beginning of good sequence data. (However, this appears to be somewhat sequence dependent since it looks like it is ~32bp for VF2 and ~25bp for VR. Stretches of a single nucleotide repeats tend to sequence badly near the beginning of a sequencing run.) Therefore, we do likely have too much spacer between the primer binding sites and the part itself in our current set of pSB plasmids. We should take this into account when determining the exact sequence near the multiple cloning site. However, if we want to ensure that the plasmid barcode is readable when sequencing with VF2 and that the plasmid barcode can be used as a primer to sequence the part, this suggests that VF2 will be some distance away from the part itself. Anyone have any thoughts on whether the barcode should be placed upstream or downstream of the part? -- RS

Barcode

Is there a plan for the barcode?

• Should the barcode only be readable by sequencing or is it sufficient to just look for an amplified band in a PCR reaction.
  • If PCR is sufficient we could build in a unique sequence just before the BB prefix and then design a reverse primer to that sequence to use along with VF.
• It seems like the most likely short-mid term problem is that a researcher would be uncertain as to which BioBrick vector they had, rather than the doomsday question of trying to work out if there is a BioBrick vector somewhere in the drink that turned Drew's hair pink.
  • Given this assumption, could we choose restriction sites, each of which are found uniquely in one of our BioBrick vectors? A researcher could just prep, digest and run on a gel to tell which vector they had.--BC
    • It might be useful to be able to tell the plasmid (and resistance) by colony PCR rather than a prep. A PCR requires less starting material. -Jkm
• There is no current plan for the barcode. The intention was just to make the identity of the plasmid obvious from a sequencing reaction but this goal is compatible with making the plasmid identifiable via a colony PCR as well. Choosing a unique restriction site for each vector would be more difficult because that would involve placing additional requirements in the BioBricks standard. i.e. Parts cannot have any of the BioBrick enzymes nor this list of restriction enzymes that are identifiers for vectors. This doesn't seem practical to me. -- RS
  • I'm not in favor of inserting restriction sites but you can probably get away without using any new enzymes under certain assumptions. First let's assume one always inserts into a new plasmid (3-way ligation, either with or without 3 antibiotic selection). Then you can just insert various combinations of BioBrick enzymes into specific locations into the plasmids and look at the pattern of bands when you cut with them. The benefit of this is let's say you cut a part with ES, run on gel, and based on the band pattern from the plasmid, you know immediately which plasmid it's in, and if it's correct, you isolate the part band and can proceed with the assembly. You have the same problem as below if one of the plasmid pieces is the same length as the part, but now you may have more potential conflicting bands. 3-antibiotic assembly without purification shouldn't really be impacted by a couple more pieces of plasmid floating around. You can also take this idea by defining another single enzyme that will be used for this purpose and you can tell plasmids apart again by the differetn lengths generated after digest. So you definitely don't need one enzyme/plasmid.

Restriction sites

• Another suggestion - Occasionally I find that I need to cut up my vector because it's of a similar size with the insert. If we could put a rare restriction site (or a couple rare restriction sites) in the middle of the backbone, that would be helpful. They could be the same restriction site(s) for each plasmid, and wouldn't have to be restrictive (you just need one that doesn't cut your part). --Jkm
  • Interesting idea but I am not sure there is a clear advantage to including a prespecified set of rare restriction enzyme sites in the plasmid. I am not sure how often this situation occurs but if it doesn't happen too often, then couldn't you just look at a restriction map of your plasmid sequence and choose an enzyme that cuts your plasmid but not your insert. It seems as if you are not necessarily buying much by prespecifying a set of sites in the plasmid cause ultimately you will always have to go in and choose a site that does not exist in your part. There are certain sites like SceI which are super long and therefore won't ever show up accidentally in a part but I would think that people might want to design parts with these extra long restriction sites in them and thus I would hesitate to put them in the plasmid. I am open to implementing this idea ... I am just not sure if it gets us very far. -- RS
  • Part of this would depend on the length of the plasmid. It's only a problem when your part and plasmid are roughly the same length. With the 1 series, though (~2kb) it came up reasonably often. I commonly use ApaLI to shred the 1 series when necessary, but every now and then it shows up in my part. It's certainly not vital - I could continue to search for a enzyme which cuts the plasmid but not the part. If I knew there was a rare enzyme (long recognition site, compatible with BB enzymes) that would work, it would make life easier. We don't even have to ban people from using that enzyme, they just need to be aware that it will be in the plasmid backbone. -Jkm
• Another idea which Austin suggested previously was to try and strip out all restriction enzymes sites from the plasmid so that they would be available for use in other applications. For instance, Austin designed the BioBricks++ assembly scheme and therefore there were some sites that he didn't want in the backbone but he only designed this new assembly scheme after many plasmids were made. And Leon stripped out a bunch of sites for the T7.1 project. However it sounds like such a task would be unhelpful to Josh unless we put some sites intentionally back in? I'm open to suggestions/comments on this idea. -- RS