Synthetic Biology:Vectors/Parts/Synthesis order

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This information is for DNA synthesis companies.

Contents

Overall design

Layout sketch of new SB vectors. Updated 8/21/2006 by Reshma Shetty.  Click to view larger version.
Layout sketch of new SB vectors. Updated 8/21/2006 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.

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: 2438 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_P1004, BBa_P1005, BBa_P1003) are modules that need to be assembled with the 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_P1004</bbpart> CmR Length: 812 Base Pairs including BioBrick ends (formerly P1000)
  • <bbpart>BBa_P1005</bbpart> TetR Length: 1326 Base Pairs including BioBrick ends (formerly P1001)
  • <bbpart>BBa_P1002</bbpart> AmpR Length: 986 Base Pairs including BioBrick ends
  • <bbpart>BBa_P1003</bbpart> KanR Length: 1010 Base Pairs including BioBrick ends

Replication origin

We want one replication origin. In addition to getting it inserted into the vector, we also want it as separate piece in BioBricks format. It is a module 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 this module (once combined with an antibiotic resistance marker) is inserted into the intact vector. Although the insertion step itself is not directional, we want clones our preferred orientation.

  • <bbpart>BBa_I50040</bbpart> pSC101 origin of replication Length: 2269 Base Pairs including BioBrick ends

Intact vector

In total, there are 3 antibiotic resistance choices * 1 replication origins = 3 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: 4500 Base Pairs
  • pSB4K5-I52000 Vector: 4698 Base Pairs
  • pSB4T5-I52000 Vector: 5014 Base Pairs

Individual BioBrick vector parts

Part number Description Size Want synthesized Want synthesized as an individual part in BioBricks format
RESISTANCE MARKERS
BBa_P1004 CmR 812bp Yes Yes
BBa_P1005 TetR 1326bp Yes Yes
BBa_P1002 AmpR 986bp Yes Yes
BBa_P1003 KanR 1010bp Yes Yes
REPLICATION ORIGINS
BBa_I50040 low copy origin (pSC101) 2269bp Yes Yes
SCAFFOLD
BBa_I51000 Vector scaffold 2438bp Yes No
COMPLETE VECTORS
pSB4C5-I52000 Vector 4500bp Yes No
pSB4K5-I52000 Vector 4698bp Yes No
pSB4T5-I52000 Vector 5014bp Yes No

BioBricks format

Certain parts (i.e. the antibiotic resistance markers and the replication origins) should be supplied separately in BioBricks format. BioBricks format means a specific 22 bp sequence upstream and a unique 21 bp sequence downstream. The upstream sequence includes an EcoRI, NotI and XbaI site. The downstream sequence includes a SpeI, NotI and PstI sequence. These sequences are included in the relevant Genbank files and labelled as BioBricks prefix and suffix.

These parts can be provided in any standard vector (like pUC19) as long as the EcoRI/SpeI/XbaI/PstI sites are unique. Alternatively, we can provide you a vector into which this part can be cloned.

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

  • Restriction sites were removed by Reshma.

Additional items for synthesis

  • Jason's screening plasmid (finished, submitted)
  • Tom's gentamycin cassette (cancelled)
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