Difference between revisions of "Part:BBa K215002"

Revision as of 05:17, 1 October 2009

pLac+RBS+Secretion Signal and Streptavidin Binding Tags

For example, assume our gene (E0040) starts with: 5'-atgcgtaaaggagaagaacttt...-3'

The design process would be:

Start with the XbaI site: 5'-TCTAGA-3'
Add ~20bp of the gene immediately after the XbaI site, starting _after_ the start codon (atg), and try to end on a G or C: 5'-TCTAGAcgtaaaggagaagaacttt-3' for E0040 from above. These ~20bp will determine the melting temperature for the primer.
Add 6-8 random nucleotides at the start. Try to balance out the primer to ~%50 g&c to a&t: 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
Tweak the number of nucleotides until the melting point roughly matches that of Vr.

Then to add the gene to the construct, again assuming the gene is a biobrick with standard suffix:

PCR with the designed forward primer and Vr
Then run the PCR product in a digest with XbaI and PstI, and digest this part (the display construct) with NheI and PstI. The XbaI site has a sticky end that binds with NheI.
Standard ligation and transformation.

Sequence and Features

Assembly Compatibility:

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K215002 short</partinfo>
-nnn
+For example, assume our gene (E0040) starts with:
+'-atgcgtaaaggagaagaacttt...-3'
+The design process would be:
+# Start with the XbaI site: 5'-TCTAGA-3'
+# Add ~20bp of the gene immediately after the XbaI site, starting _after_ the start codon (atg), and try to end on a G or C: 5'-TCTAGAcgtaaaggagaagaacttt-3' for E0040 from above.  These ~20bp will determine the melting temperature for the primer.
+# Add 6-8 random nucleotides at the start.  Try to balance out the primer to ~%50 g&c to a&t: 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
+# Tweak the number of nucleotides until the melting point roughly matches that of Vr.
+Then to add the gene to the construct, again assuming the gene is a biobrick with standard suffix:
+# PCR with the designed forward primer and Vr
+# Then run the PCR product in a digest with XbaI and PstI, and digest this part (the display construct) with NheI and PstI.  The XbaI site has a sticky end that binds with NheI.
+# Standard ligation and transformation.
 <!-- Add more about the biology of this part here