Synthetic Biology:Vectors/pSB**5 design
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Revision as of 23:01, 12 July 2006
These are some design notes on the new version 5 vectors.
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).
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
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)
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|
|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)|
|BBa_I50000||F plasmid origin||4640bp||Yes||Yes|
|BBa_I50040||low copy origin (pSC101)||2215bp||Yes||Yes|
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
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.
- 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.
- We may want to include a unique cutter in the scaffold vector. This has not been included yet since the sequences are not in final form. Once the sequences of the coding regions are closer to being settled, I will add a 6bp unique cutter to the backbone.
- Likely want a unique cutter in the backbone, in each origin and each resistance marker if possible.