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.

One plan that I am currently considering is actually encoding the name of the plasmid in DNA. For instance,

AAA = 0; AAC = 1; AAG = 2; AAT = 3; . . . AGC = 9; AGG = A; AGT = B; . . . GAT = Z;

So that you could literally write out pSB5AC4-P1010.I50020 in DNA. Of course, we may want to make this slightly more intelligent to space out characters, include start and stop strings and avoid key codons like ATG, TAA and TGA. Any comments? --RS

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

Enabling vector DNA purification

• Use a secondary, inducible copy number origin or insert the pUC backbone inside of the BioBricks cloning site to facilitate prepping of the vector?
  • Inducible copy number origin
    • The most common system for inducible copy number F-based plasmid requires a special strain (a copy up mutant of traF under the control of an arabinose inducible promoter) for inducible behavior.
    • The plasmid should operate at single copy in most other strains.
    • Systems containing the arabinose promoter would not be able to be induced to higher copy in the special strain without also affecting system behavior.
    • This option has the advantage that parts can be prepped from the F plasmid.
    • Could not easily make use of ccdB as a selection marker because DB3.1 does not express the traF gene necessary for expression at high copy. sacB is an alternative but requires sucrose in the media.
      • I must not be understanding this, isn't DB3.1 resistant to "ccdB" and hence you wouldn't be using DB3.1 for selection anyway?
        • I was referring to the fact that prepping the plasmid for use would be difficult. I envision using this plasmid not to assemble BioBrick parts but as the vector in which characterization takes place. Therefore, I assume we would more often want to prep the vector (which contains ccdB) than a vector with a part in it. Thus, in order to easily prep large amounts of vector, we would need a host strain that can tolerate ccdB and has the traF copy up mutation which likely does not exist (and therefore would need to be constructed). I am not convinced that we will want to prep parts in the F plasmid so frequently that it is worth the effort of constructing such a strain. -- RS
  • pUC19 origin in the BioBricks insertion site
    • simply inserting a pUC backbone into the BioBricks insertion site enables the plasmid to be prepped easily and does not introduce any incompatibility issues.
    • allows the plasmid to be prepped at very high copy
    • parts in the F plasmid cannot be easily prepped
      • So this means we'd have to TempliPhi in order to sequence? The TempliPhi website seems to think this is reliable enough to work--BC
      • Sequencing could be done by either sequencing a PCR product or via Templiphi. You can prep F plasmid from cells, it is just not quite as simple and quick as a miniprep since you get much lower yields. -- RS

Other discussion

• Are we sure that F plasmids are really at 1-2 copies per cell? Why was pSB2K3-1 measured to be higher than that?
  • From Johann Paulsson: it is unclear how tight of control F plasmid based vectors have over copy number fluctuation. Having the vector exist at single copy strongly depends on generation time. Faster growing cells are more likely to have multiple overlapping rounds of replications occurring simultaneously.
• What parts of the F plasmid are responsible for integration onto the genome? Can we omit them?
  • cos and/or loxP sites are generally used for integration in the genome. Currently, I have no plans to include them in this vector.
• Many of the existing BACs only seem to have a partial sopC CDS, do we want the rest?
  • pSMART VC vector appears to have a more complete sopC region. This may lead to tighter control of copy number.
• A set of orthogonal single copy replication origins to allow multiple vectors to be maintained in a cell. Can we have a set of vectors with F and P1 origins?--BC 17:36, 31 Oct 2005 (EST)
  • Not sure this is possible. I believe the P1 origins use the par set of genes to maintain single copy whereas the F origins use the sop set of genes. The two sets are pretty homologous to eachother and therefore likely incompatible. I need to check on this more. -- RS
  • Perhaps derivatives from the two plasmids the Berkeley iGEM team used might permit two single copy vectors to be used simultaneously. -- RS
• Should the flanking terminators be placed outside the VF2 and VR primer binding sites? Is it useful to have them within? Moving the flanking terminators outside the primer binding sites means fewer bases to sequence through before hitting the part. Alternatively, we could move the terminators to just inside the primers since anyway ~40bp are needed before sequence data is of high quality.
  • Tom thinks this doesn't matter but suggests including some translational stops around the multiple cloning site

• Choose between manual assembly of vector modules or direct synthesis of all plasmid variants
  • Can we get a price break for synthesizing multiple plasmid variants?
  • How many assemblies would we need to do?
  • Is there a hybrid approach? Could we PCR the F plasmid backbone since its long and then have the collection of smaller parts (that would otherwise involve several assemblies) synthesized? Maybe a partial synthesis approach would help get around the issue of constructing a BioBricks insertion site?--BC
    • PCR'ing the F plasmid backbone is not very practical since there are several BioBricks sites in the backbone each of which would need to be individually mutated out. It is unlikely that there is anyone who is willing to do this much work. Therefore, the current plan is to synthesize the backbone. -- RS
• If all the vector components are specified in BioBricks format, how do we construct a BioBricks insertion site?
  • Blunt-end ligation?
  • Other restriction enzyme sites?
  • PCR
  • Use special restriction sites for vector construction (Austin's idea). Expanding on this, we could define a new idempotent assembly standard for exclusive use for vector components.
• Should we reduce the spacing between the verification primer binding sites and the BioBricks site to 30bp? This may require human intervention to read sequence at around the 60 bp mark. Such a change requires that the flanking terminators be moved outside the verification primer binding sites.
• Should we allow swapping of just one of the resistance markers (i.e. cmR/tetR/kanR) or both (i.e. ampR + another)?
• One risk of permitting the two antibiotic resistance markers to transcribe in opposite directions is that antisense RNA is created to the other resistance marker, rendering it useless. Thus, the resistance markers should perhaps point in the same direction away from the multiple cloning site. Therefore, both resistance markers should likely be on the opposite strand (which would mean they would need to be remade via PCR). Or instead, the origins could be put on the opposite strand.