Team:BGU Israel/Parts
Link: PR1 Promoter link
The PR1 promoter is an attenuated PR promoter from the pBEST plasmid backbone. Previous to our project, this modified promoter was designed in our lab, placing a point mutation that slows down the initial translation initiation rates, resulting in better in-vivo protein translation.
We used this promoter in all four of our polycistronic plasmid designs and showed the use of this promoter in polycistronic devices. In one of our plasmids (BBa_K2938016) we were able to show the translation of three genes downstream of this promoter.
For each of our polycistronic plasmids we placed a deGFP marker downstream of the rest of the genes, and near the terminator. The fluorescence of the GFP is an indication that the upstream genes (the toxin subunits) were also translated.
In the following table you can see fluorescence in all Serratia bacteria expressing our polycistronic plasmids [BBa_K2938014 - Cry4Ba, BBa_K2938015 - Cry11Aa, BBa_K2938016 - Cry11Aa + p20] compared to the same bacteria expressing a plasmid containing only a deGFP gene (GFP) and a wild-type bacteria, without a deGFP gene.
Other Basic parts we added:
In our project we used the Bacillus thuringiensis subsp. israelensis (Bti) toxin subunits.
Some of these toxin genes were used by previous iGEM teams and projects, but had various point mutations throughout the DNA sequence compared to the published sequences. That is why we added these basic parts again.
Also, we designed and added commonly used tags (HA, Strep and His) to our subunits in order to be able to detect them using Western Blot analysis using anti-tags antibodies.
The parts containing the tags appear in our designed plasmids (devices) that we used and presented during our project.
We also found some changes between our tags and tags that were already in the registry, so we added our versions of the tags. Also, we added the terminator sequence that we used in all of our devices.
| Cry11Aa | Cry11Aa - No tag | Cry11Aa - Strep tag |
| Cry4Ba | Cry4Ba - No tag | Cry4Ba - HA tag |
| Cyt1Aa | Cyt1Aa - No tag | Cyt1Aa - Strep tag |
| P20 | P20 - No tag | P20 - His tag |
| Strep tag | Strep tag |
| His tag | His tag |
| T500 Terminator | T500 Terminator |