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
  • 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