Difference between revisions of "Team:DTU-Denmark/Design Promoter"
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We have designed the LEAP promoters not just to be compatible with the new iGEM Type IIs RFC[1000] standard, but with a range of other assembly standards as well. The domestication of promoters was done by making a prioritized list of standards to be compatible with. Using this prioritized list, the scoring system seen in table 01 was implemented into the software used to design the promoters. <br> | We have designed the LEAP promoters not just to be compatible with the new iGEM Type IIs RFC[1000] standard, but with a range of other assembly standards as well. The domestication of promoters was done by making a prioritized list of standards to be compatible with. Using this prioritized list, the scoring system seen in table 01 was implemented into the software used to design the promoters. <br> | ||
| − | This scoring system resulted in a large number of the promoters in the final promoter library being compatible with some of the most widely used Type IIs standards, as seen under the design considerations sections on the pages of their respective parts. | + | This scoring system resulted in a large number of the promoters in the final promoter library being compatible with some of the most widely used Type IIs standards, as seen under the design considerations sections on the pages of their respective parts. <br> |
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Revision as of 14:01, 21 October 2019
Promoter Design
Promoter design considerations
Our software was designed to produce promoters that would be compatible with a large range of cloning systems and be easy to de novo synthesize.
We have designed the LEAP promoters not just to be compatible with the new iGEM Type IIs RFC[1000] standard, but with a range of other assembly standards as well. The domestication of promoters was done by making a prioritized list of standards to be compatible with. Using this prioritized list, the scoring system seen in table 01 was implemented into the software used to design the promoters.
This scoring system resulted in a large number of the promoters in the final promoter library being compatible with some of the most widely used Type IIs standards, as seen under the design considerations sections on the pages of their respective parts.
| Enzyme/Cloning system | Score | Justification |
|---|---|---|
| SwaI enzyme | 2 | SwaI domestication can be used for linearisation of vectors for genomic integration. This is something we have considered working on in the near future |
| MoClo | 1.5 | MoClo (level 0) is **** |
| Mobius | 1 | |
| GoldenBraid | 0.3 | |
| Chi | 0.2 |
For comparison, we have analysed a range of 15 different native promoters from Aspergillus spp. genes that our model has based the synthetic promoters upon. For 8 of those native promoters, we found one or more Type IIs sites from either BsaI or SapI, making these native promoters incompatible with the RFC[1000] standard. Domestication of these native promoters is not trivial, as disruption of any transcription factor binding sites when changing the nucleotide sequence of the promoter. By including the domestication of the synthetic promoters in the design from the very beginning, we are producing the best performing promoters that are still compatible with the different assembly standards.
We have also designed the software to produce promoters that are easily synthesizable by de novo synthesis. This was done by creating some design rules with the algorithm that would keep the global GC% content of the promoters within a certain threshold (For the LEAP promoters, the threshold was set at between 25% and 65% GC content), as well as prevent the promoters from having A/T or G/C homopolymers. This threshold can be changed depending on the constraints of the specific situation.
Differentiating between phases
With the design rules for the synthetic promoters described above, it was possible to design promoters with different characteristics. According to our feedback from Novozymes and Zymergen, a useful promoter characteristic for large scale productions is the ability to have a low gene expression during exponential growth while having a strong gene expression in stationary phase. This inspired us to look for promoters with different dynamics, that would be active under different phases of cell growth. By looking at RNA-seq data, we were able to identify genes that had low activity in the exponential phase and high activity in the stationary phase, and vice versa for additional promoter coverage. We expected the synthetic promoters based on these selected genes to be active during the same phases of cell growth as the native genes they were based on.
| Gene name (systematic) | Reasoning |
|---|---|
| mstA | Should be high constitutive but regulated by sugar availability |
| gpdA | A strong constitutive promoter that is commonly used in industry |
| glaA | High activity in exponential phase, low activity in stationary phase |
| hfbD | Should be most active in stationary phase |
Sources here will also come soon
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