Source:
Generated By: https://synbiohub.org/public/igem/igem2sbol/1
Created by: Alex Leone
Date created: 2009-09-29 11:00:00
Date modified: 2015-05-08 01:11:30
Generic Surface Display Construct: IPTG Inducible Lpp + 1tmr OmpA + NheI Site
| Types | DnaRegion |
| Roles | Generator engineered_region |
| Sequences | BBa_K215200_sequence (Version 1) |
Description
This is a generic surface display construct that displays a protein with the OmpA surface display system as described in [cite].To insert a gene, you will need to design a primer that removes the start codon and adds a XbaI site that keeps the rest of the gene in frame with OmpA. This assumes that the gene is a biobrick with the standard suffix following the gene. If you are creating the gene from scratch, biobrick it then follow this guide so that there is the standard suffix following the gene.
For example, assume our gene (E0040) starts with:
5'-atgcgtaaaggagaagaacttt...-3'
The design process would be:
1. Start with the XbaI site: 5'-TCTAGA-3'
2. 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.
3. Add 6-8 random nucleotides at the start. Try to balance out the primer to ~%50 g&c to a&t: 5'-cgggcTCTAGAcgtaaaggagaagaacttt-3'
4. 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:
1. PCR with the designed forward primer and Vr
2. 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.
3. Standard ligation and transformation.
Notes
We took the Lpp Signaling Peptide and OmpA 1 trans-membrane region from Havard 2006, BBa_J36848, with two custom primers:Lpp Forward: 5'-gcggccgctTCTAGAtgaaagctactaaactggtactggg-3'
OmpA Reverse with GS Linker, GGGSGGGSGGG, and a TEV site, ENLYFQG, and SpeI site: 5'-cggccgctACTAGTaGCTAGCaccctgaaaatacaggttttcACCACCACCAGAACCACCACCAGAACCACCACCgctgcctttgtacggcatacgacc-3'
Source
The Lpp + 1tmr OmpA is from Havard iGEM 2006: BBa_J36848, which is from [cite].| Access | Instance | Definition |
| public public public | BBa_R0011 BBa_B0034 BBa_K215250 | lacI+pL BBa_B0034 BBa_K215250 |
| Sequence Annotation | Location | Component / Role(s) |
| BBa_R0011 BBa_B0034 BBa_K215250 | 1,54 64,75 82,297 | lacI+pL BBa_B0034 BBa_K215250 |