Difference between revisions of "Part:BBa K799026"

Revision as of 03:22, 3 October 2012

pSB1C3 yGG transcriptional terminator acceptor vector

The original pSB1C3 backbone has been adapted so that yeast Golden Gate (yGG) TT (transcriptional terminator) parts, conforming to RFC88, can be quickly converted into BioBrick parts that are compatible with the BioBrick assembly standard.

Between the BioBrick prefix and suffix we have introduced two recognition sequences for the type IIS restriction enzyme BsaI. The BsaI sites flank an RFP gene and are oriented to eliminate themselves and the RFP during digestion and ligation of a yGG TT part. The 4 base pair sticky ends generated by BsaI digestion of the modified pSB1C3 vector are complementary to the 4 base pair overhangs in our Yeast Golden Gate TT parts, thus enabling yGG to BioBrick TT part conversion.

The best way to use this part is to perform a simultaneous digestion-ligation reaction, whereby the yGG TT construct, the modified pSB1C3 vector, BsaI, and T4 DNA ligase are all introduced into a single tube and incubated at 30C for 1 hour, 50C for 5 minutes, and 80C for 5 minutes. Following E.coli transformation, selection on chloramphenicol plates will entirely eliminate the background of cells transformed with the original yGG promoter construct. Newly assembled constructs, in which the yGG TT part has been converted into a BioBrick part, will make white colonies compared to the red colonies produced by intact, RFP-containing pSB1C3 vector.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal XhoI site found at 1012
Illegal XhoI site found at 1904
23
INCOMPATIBLE WITH RFC[23]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
25
INCOMPATIBLE WITH RFC[25]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.

Plasmid Map

Sequencing

[[]]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K799026 short</partinfo>
-The original psb1c3 backbone was modified in order to allow for Golden Gate enabled parts to easily become biobricks. Within the iGEM prefix and suffix, extra overhangs were added containing the appropriate Bsa1 sites for Golden Gate Assembly.
+The original pSB1C3 backbone has been adapted so that yeast Golden Gate (yGG) TT (transcriptional terminator) parts, conforming to RFC88, can be quickly converted into BioBrick parts that are compatible with the BioBrick assembly standard.
+Between the BioBrick prefix and suffix we have introduced two recognition sequences for the type IIS restriction enzyme BsaI. The BsaI sites flank an RFP gene and are oriented to eliminate themselves and the RFP during digestion and ligation of a yGG TT part. The 4 base pair sticky ends generated by BsaI digestion of the modified pSB1C3 vector are complementary to the 4 base pair overhangs in our Yeast Golden Gate TT parts, thus enabling yGG to BioBrick TT part conversion.
+The best way to use this part is to perform a simultaneous digestion-ligation reaction, whereby the yGG TT construct, the modified pSB1C3 vector, BsaI, and T4 DNA ligase are all introduced into a single tube and incubated at 30C for 1 hour, 50C for 5 minutes, and 80C for 5 minutes. Following E.coli transformation, selection on chloramphenicol plates will entirely eliminate the background of cells transformed with the original yGG promoter construct. Newly assembled constructs, in which the yGG TT part has been converted into a BioBrick part, will make white colonies compared to the red colonies produced by intact, RFP-containing pSB1C3 vector.
 <!-- Add more about the biology of this part here