Difference between revisions of "Part:pSB1A10:Design"

Revision as of 01:49, 31 May 2006

The process for building pSB1A10,was:

Build I13534 using normal BB construcion.
Insert I13534 into psb1A3 plasmid
Cap the BB prefix and suffix using I13450
Re-insert a BB prefix/suffix into the center of I13534 using primers to the "insertion region" of I13457 and around-the-horn PCR.

Some design notes:

The inclusion of the 'barcode' was unintentional (it was attached to E1010), it has no (intended) function.
The "spacer artifacts" at position 22 and 5225 are artifacts from around-the-horn PCR for inserting the BB restriction sites into the center of the plasmid. If you look at I13457 you will see the corresponding regions in the section annotated "insertion region".
The RNAse E sites are intended to insulate the device being screened from the screening plasmid itself. See a more detailed explanation [http://openwetware.org/wiki/Endy:Screening_plasmid_1.0 here].
The hairpin 5' to E1010 is meant to stabilize the mRNA transcript by preventing degredation by RNAse E. <site>keasling</site>
- however, there is a C deletion at bp 64 from the sequence used previouslykeasling. For our current purposes the expression level of E1010 (mRFP1) has been sufficient with this mutated form of the hairpin, so we haven't made an effort to change it.

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@@ Line 2: / Line 2: @@
 #Build [[Part:BBa_I13534|I13534]] using normal BB construcion.
 #Insert [[Part:BBa_I13534|I13534]] into psb1A3 plasmid
-#Scar out the BB prefix and suffix using [[Part:BBa_I13450|I13450]]
+#Cap the BB prefix and suffix using [[Part:BBa_I13450|I13450]]
 #Re-insert a BB prefix/suffix into the center of [[Part:BBa_I13534|I13534]] using primers to the "insertion region" of [[Part:BBa_I13457|I13457]] and around-the-horn PCR.