Synthetic Biology:Vectors/Parts

From OpenWetWare
Jump to navigationJump to search

This is a list of BioBricks parts for use in construction of modular vectors.

Some have just been designed while others have been constructed and tested. See the associated registry page for details.

Replication origins

  • <bbpart>BBa_I50000</bbpart>: F plasmid backbone with BioBricks restriction sites removed.
  • <bbpart>BBa_I50001</bbpart>: F plasmid backbone with BioBricks restriction sites removed, reverse orientation.
  • <bbpart>BBa_I50010</bbpart>: oriV origin which requires TrfA protein to be functional.
  • <bbpart>BBa_I50020</bbpart>: high copy origin of replication from pSB1A3
  • <bbpart>BBa_I50030</bbpart>: a pBR322 origin.
  • <bbpart>BBa_I50040</bbpart>: a near minimal pSC101 origin.
  • <bbpart>BBa_I50041</bbpart>: a near minimal pSC101 origin, reverse orientation.
  • <bbpart>BBa_I50050</bbpart>: a R6K gamma origin. See also EZ-Tn5™ <R6Kγori /KAN-2> Sequence general information (Note: this origin will only replicate in a pir+ strain.)

Antibiotic resistance cassettes

  • <bbpart>BBa_P1001</bbpart>: cassette providing tetracycline resistance.
  • <bbpart>BBa_P1000</bbpart>: cassette providing chloramphenicol resistance.
  • <bbpart>BBa_P1002</bbpart>: cassette providing ampicillin resistance.
  • <bbpart>BBa_P1003</bbpart>: cassette providing kanamycin resistance.
  • <bbpart>BBa_P1004</bbpart>: cassette providing ampicillin resistance in reverse orientation.
  • <bbpart>BBa_P1005</bbpart>: cassette providing tetracycline resistance and ampicillin resistance with terminators.
  • <bbpart>BBa_P1006</bbpart>: cassette providing chloramphenicol resistance and ampicillin resistance with terminators.
  • <bbpart>BBa_P1007</bbpart>: cassette providing kanamycin resistance and ampicillin resistance with terminators.

Terminators

  • <bbpart>BBa_B0055</bbpart>: upstream flanking terminator
  • <bbpart>BBa_B0054</bbpart>: downstream flanking terminator
  • <bbpart>BBa_B0053</bbpart>: bidirectional terminator from E. coli his operon
  • <bbpart>BBa_B0052</bbpart>: forward terminator
  • <bbpart>BBa_B0062</bbpart>: reverse terminator of BBa_B0052.

Notes

It wasn't clear from the website if these were bi-directional? Not sure that this is very important--BC

I don't know if the terminators are bidirectional. These terminators are used as flanking terminators in other vectors and are claimed to make the cloning of difficult pieces of DNA (like strong promoters) easier. This is why I was planning on orienting the antibiotic resistance cassette in the opposite direction so that read through from upstream of the multiple cloning site is less of an issue. -- RS

Primer binding sites

  • <bbpart>BBa_G00100</bbpart>: VF2
  • <bbpart>BBa_G00102</bbpart>: VR

Others

Minimal vector scaffold

5' <counter-selectable marker> -- <high copy origin> -- BBa_G00001 (BBsuffix) -- BBa_B0044 (TOPO site) -- BBa_B0042 (translational stop sequence) -- <plasmid barcode> -- BBa_B0054 (terminator) -- BBa_G00102 (VR) -- BBa_B0045 (antibiotic resistance insertion site) -- BBa_B0046 (origin insertion site) -- BBa_G00100 (VF2) -- BBa_B0055 (terminator) -- <plasmid barcode> -- BBa_B0042 (translational stop sequence) -- BBa_B0043 (TOPO site) -- BBa_G00000 (BB prefix) 3'

Removal of restriction sites

Given that the current plan is to synthesize the vectors, we can remove restriction sites, mostly at will from the vectors. What restriction sites should be removed?

BioBrick enzymes sites

EcoRI, SpeI, XbaI, PstI, NotI

Enzymes sites that generate compatible cohesive ends to BioBrick sites

Offset cutters

AarI, BsmBI, BsaI, BbsI, BspMI, BtgZI

Other common enzymes

Arbitrary list, feel free to add more.

HindIII, BamHI, XhoI, NcoI, SacI, NdeI,

Would be simplest to destroy all 6 bp palindromic sequences. This destroys a lot of the common 'normal' restriction sites.

  • Is there a tool that does this? GeneDesign removes sites and optimizes the resulting codons for a particular species. But it requires that you select which enzymes you want to remove from their list.
  • Don't know off the top of my head but you should ask Sri or Leon if there's a tool. They did that for their plasmid for the T7.1 rebuild so they could use more restriction enzymes. GeneDesign appears to actually list the enzyme sites that are in the sequence so assuming it has a good database of enzymes, the question is whether just removing everything it knows about is good enough. I could pretty easily write a program to find all 6 bp palidromes but fixing it with silent mutations would take more work.

GATC

Another idea I had was whether we want to remove all GATC (DpnI) sites from the plasmid. There are potential benefits and drawbacks to this. One potential downside is that some mutation protocols assume that you can chew up the plasmid by adding DpnI. However if our plasmid had no GATC, we could perhaps use this to our advantage in some way. For example, adding DpnI to cut up only the genomic DNA and not our plasmid (of course, this would only likely work with the base plasmids).

  • RS 11:55, 15 May 2006 (EDT): Tom actually requested that I specifically include GATC sites in the plasmid to ensure that digestion by DpnI works well. For cloning purposes, I think that treatment of the destination plasmid digestion with antarctic phosphatase is generally sufficient for reducing the likelihood of cloning genomic DNA. So I would favor that approach over removing DpnI sites (given that the presence of DpnI sites is useful for site-directed mutagenesis).

Ordering information

Independently synthesized components

(This information is for DNA synthesis companies).

Vector scaffold

><bbpart>BBa_I51000</bbpart> Minimal vector scaffold Length: 408 Base Pairs Including Part and Barcode Circular

This sequence is the vector scaffold.

Antibiotic resistance marker

We want four different resistance markers. For these parts, in addition to getting them inserted into the vector, we'd also want them as separate pieces in BioBricks format. There is flexibility in the coding sequence of these parts. One of them, BBa_P1002 is present in the vector scaffold but we would also like it as a separate piece in BioBricks format. The other three (BBa_P1000, BBa_P1001, BBa_P1003) are modules that need to be assembled with each replication origin and then inserted at the two NheI restriction sites in the vector scaffold (to replace BBa_P1002).

  • <bbpart>BBa_P1000</bbpart> CmR Length: 835 Base Pairs including BioBrick ends
  • <bbpart>BBa_P1001</bbpart> TetR Length: 1322 Base Pairs including BioBrick ends
  • <bbpart>BBa_P1002</bbpart> AmpR Length: 984 Base Pairs including BioBrick ends
  • <bbpart>BBa_P1003</bbpart> KanR Length: 1036 Base Pairs including BioBrick ends

Replication origin

We want two different replication origins. For these parts, in addition to getting them inserted into the vector, we'd also want them as separate pieces in BioBricks format. There is flexibility in the coding sequence of these parts. Note the orientation in which these two replication origins are inserted into the intact vector.

  • <bbpart>BBa_I50000</bbpart> F plasmid backbone with BioBrick sites removed Length: 4683 Base Pairs including BioBrick ends
  • <bbpart>BBa_I50040</bbpart> pSC101 origin of replication Length: 2258 Base Pairs including BioBrick ends

Insert

Note that BBa_P1011 and BBa_I52000 encode ccdB, a toxic gene to E. coli, and therefore will only propagate in special strains which have a mutation that confers resistance to ccdB (i.e. DB3.1). BBa_I52000 is the insert present in the vector scaffold. Depending on cost, we may or may not ask for the BBa_I50020 and BBa_P1011 as separate pieces in BioBricks format.

  • <bbpart>BBa_P1011</bbpart> ccdB cassette Length: 485 Base Pairs including BioBrick ends
  • <bbpart>BBa_I50020</bbpart> High copy origin of replication Length: 861 Base Pairs including BioBrick ends
  • <bbpart>BBa_I52000</bbpart> ccd with high copy origin Length: 1303 Base Pairs including BioBrick ends (without a scar)

Intact vector

In total, there are 3 antibiotic resistance choices * 2 replication origins = 6 total intact vector combinations.

This is one out of the six possible intact vectors that we would like to get synthesized. It has the insert, both BBa_P1000 and BBa_P1008 as the antibiotic resistance markers and BBa_I50001 origin in the appropriate locations. We would want this returned as circular DNA.

><bbpart>pSB5AC4</bbpart> pSB5AC4 Length: 8257 Base Pairs Including Part and Barcode

Individual BioBrick vector parts

Part number Description Size BioBrick available Template available Want synthesized as an individual part in BioBricks format
INSERT
BBa_P1011 ccdB 442bp No No Maybe
BBa_I50020 high copy origin (pUC19) 818bp No Yes Maybe
BBa_I52000 ccdB and high copy origin (pUC19) 1260bp No No No (this is part of the vector scaffold)
RESISTANCE MARKERS
BBa_P1000 CmR 792bp No No Yes
BBa_P1001 TetR 1279bp No No Yes
BBa_P1002 AmpR 941bp No No Yes
BBa_P1003 KanR 993bp No No Yes
REPLICATION ORIGINS
BBa_I50000 F plasmid origin 4640bp No No Yes
BBa_I50040 low copy origin (pSC101) 2215bp No No Yes
Retrieved from "https://openwetware.org/mediawiki/index.php?title=Synthetic_Biology:Vectors/Parts&oldid=40771"