Synthetic Biology:Vectors/pSB**5 design

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(Unique restriction sites in the backbone)
(Offset cutters)
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===Offset cutters===
===Offset cutters===
[http://www.fermentas.com/catalog/re/aari.htm AarI], [http://www.neb.com/nebecomm/products/productR0580.asp BsmBI], [http://www.neb.com/nebecomm/products/productR0535.asp BsaI], [http://www.neb.com/nebecomm/products/productR0539.asp BbsI], [http://www.neb.com/nebecomm/products/productR0502.asp BspMI], [http://www.neb.com/nebecomm/products/productR0703.asp BtgZI],
[http://www.fermentas.com/catalog/re/aari.htm AarI], [http://www.neb.com/nebecomm/products/productR0580.asp BsmBI], [http://www.neb.com/nebecomm/products/productR0535.asp BsaI], [http://www.neb.com/nebecomm/products/productR0539.asp BbsI], [http://www.neb.com/nebecomm/products/productR0502.asp BspMI], [http://www.neb.com/nebecomm/products/productR0703.asp BtgZI],
-
[http://www.neb.com/nebecomm/products/productR0528.asp EarI],  
+
[http://www.neb.com/nebecomm/products/productR0528.asp EarI], BbvCI
===Nicking enzymes===
===Nicking enzymes===

Revision as of 18:29, 14 July 2006

These are some design notes on the new version 5 vectors.

Contents

Overall design

Layout sketch of new SB vectors. Updated 6/19/2006 by Reshma Shetty.  Click to view larger version.
Layout sketch of new SB vectors. Updated 6/19/2006 by Reshma Shetty. Click to view larger version.


Vector components

Genbank files of the sequences below are available for download.

This is the list of DNA fragments that need to be synthesized (with the exception of the Insert which comes as part of the scaffold).

Vector scaffold

This sequence is the vector scaffold. It has BBa_P1011 (ccdB, a toxic gene) and BBa_I50020 (pUC19 origin) in the multiple cloning site. It also has BBa_P1002 (ampicillin resistance cassette in the reverse orientation) flanked by two NheI sites. Digestion of this plasmid with NheI will permit any BioBrick part that has been cut with XbaI and Spe I to be inserted into the vector.

  • <bbpart>BBa_I51000</bbpart> Vector scaffold Length: 2606 Base Pairs

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). Note the orientation in which these modules (once combined with a replication origin) are inserted into the intact vector. Although the insertion step itself is not directional, we want clones in our preferred orientation.

  • <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. These are modules that need to be assembled with each antibiotic resistance marker and then inserted at the two NheI restriction sites in the vector scaffold (to replace BBa_P1002). Note the orientation in which these modules (once combined with an antibiotic resistance marker) are inserted into the intact vector. Although the insertion step itself is not directional, we want clones our preferred orientation.

  • <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 (or MCS module)

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. We likely will 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 from pUC19 Length: 861 Base Pairs including BioBrick ends
  • <bbpart>BBa_I52000</bbpart> MCS cassette, ccdB 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. The antibiotic resistance markers and replication origins have been assembled as a unit and inserted into the vector scaffold at the NheI sites. Note that this cloning step is not directional although we do want the replication origin and antibiotic resistance marker to be inserted into the vector in a particular orientation. Therefore, this cloning step will require some screening to verify orientation.

  • pSB4C5-I52000 Vector: 4682 Base Pairs
  • pSB4K5-I52000 Vector: 4883 Base Pairs
  • pSB4T5-I52000 Vector: 5169 Base Pairs
  • pSB5C5-I52000 Vector: 7107 Base Pairs
  • pSB5K5-I52000 Vector: 7308 Base Pairs
  • pSB5T5-I52000 Vector: 7594 Base Pairs

Individual BioBrick vector parts

Part number Description Size Want synthesized Want synthesized as an individual part in BioBricks format
INSERT
BBa_P1011 ccdB 442bp Yes Maybe
BBa_I50020 high copy origin (pUC19) 818bp Yes Maybe
BBa_I52000 ccdB and high copy origin (pUC19) 1260bp Yes No (this is part of the vector scaffold)
RESISTANCE MARKERS
BBa_P1000 CmR 792bp Yes Yes
BBa_P1001 TetR 1279bp Yes Yes
BBa_P1002 AmpR 941bp Yes Yes
BBa_P1003 KanR 993bp Yes Yes
REPLICATION ORIGINS
BBa_I50000 F plasmid origin 4640bp Yes Yes
BBa_I50040 low copy origin (pSC101) 2215bp Yes Yes
SCAFFOLD
BBa_I51000 Vector scaffold 2606bp Yes No
COMPLETE VECTORS
pSB4C5-I52000 Vector 4682bp Yes No
pSB4K5-I52000 Vector 4883bp Yes No
pSB4T5-I52000 Vector 5169bp Yes No
pSB5C5-I52000 Vector 7107bp Yes No
pSB5K5-I52000 Vector 7308bp Yes No
pSB5T5-I52000 Vector 7594bp Yes No

BioBricks format

The antibiotic resistance markers and the replication origins will be supplied separately in BioBricks format.

These parts will be provided in a standard vector (like pUC19).

Vector assembly scheme

Sample assembly scheme for vectors.  The same scheme can be used for all combinations of replication origins and antibiotic resistance markers.  Click to view larger version.
Sample assembly scheme for vectors. The same scheme can be used for all combinations of replication origins and antibiotic resistance markers. Click to view larger version.


Restriction enzyme site removal

  • Tried to remove the suggested restriction sites from the vector components whenever possible. Some sites which occurred in non-coding regions (like nicking enzymes and ApoI sites) were not removed.

Here are the restriction enzyme sites that I tried to remove.

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, EarI, BbvCI

Nicking enzymes

Nt.BstNBI

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).
  • Reshma 15:49, 14 July 2006 (EDT): Note that I have left the GATC sites.

Unique restriction sites in the backbone

Sites for barcode insertion

Drew wanted a unique restriction site in the backbone via which a barcode could be inserted at a later date. Austin argues that we shouldn't arbitrarily include 6bp cutters in the backbone because they could be used in parts.

Barcodes can be inserted easily into future vectors by simply assembling them with an origin and resistance marker in BioBricks format and inserting them into the NheI sites in the vector scaffold. But what about the intact vectors we're ordering?

Comments welcome.

Sites to cut vector backbone

Josh initially requested unique cutters in the backbone so that in the event that the BioBrick part is the same size as the vector backbone, you could cut the backbone to enable size separation of the two.

Austin suggested using BaeI ... a so-called useless enzyme because there is some uncertainty in exactly where it cuts. BaeI already cuts in the middle of <bbpart>BBa_I50000</bbpart> but does not cut <bbpart>BBa_I50040</bbpart>. I'll introduce a BaeI cut site in BBa_I50040 and this enzyme will serve as the backbone cutter.

Additional features

  1. How much would it cost to include 4 unique bases adjacent to VF2 and VR primer binding sites (present in the scaffold)? These 4 unique bases should uniquely identify the vector. What would this add to the cost?
    • Inclusion of these bases seems to add about $6000 to the cost of the project therefore we are not including barcodes at this time.
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