Team:UIUC Illinois/Design
Design Overview
Based on a paper we found, we found 2 genes that could help us degrade glyphosate in E. coli (L Herrera-Estrella, 1995).
Parts:
- glpA: A gene that codes for a phosphoryl transferase, that phosphorylates glyphosate, making it less toxic to the cell. This in turn increases cellular resistance to glyphosate. Also referred to as hph
- glpB: Codes for glyphosate oxidoreductase, and breaks down glyphosate to AMPA by cleaving the C-N bond on glyphosate. Also referred to as C-N lyase or simply lyase. The diagram below provides a basic overview of how our construct would work:
The glyphosate oxidoreductase (glpB) would cleave the C-N bond on glyphosate, resulting in the formation of a common degradation product of glyphosate often seen in the environment, aminomethylphosphonic acid (AMPA). AMPA would then be further broken down into harmless products such as methylamine via E. coli's native C-P lyase.
In our construct, we attached different promoters to each of our genes to provide some versatility in expression levels. This would also allow us to test the individual efficacy of our genes on degradation of glyphosate.
Using iGEM parts, this is how we designed our construct:
Restriction Digestion
The first design iteration we went through was constructing our plasmid using restriction digestion, and basic BioBrick cloning. We ordered the genes from Integrated DNA Technologies (IDT) individually with the iGEM prefix and suffix. This allowed us to potentially combine our genes in whichever order we wanted as well as with any promoters that were provided as part of the DNA Distribution Kit.
We attempted to first combine our glpB gene with a plasmid containing pLac + RBS. We ordered pTet + RBS as a separate gBlock since the RBS was too small to purify via Miniprep or any other methods we had access to. We aimed to combine glpA with the pTet + RBS gBlock outside a plasmid and then combine it with our plasmid containing pLac, RBS and glpB. Unfortunately, we ran into several problems while attempting to create our various parts and the plasmid.
Our restriction digestions didn’t seem to be working as expected, so we ran multiple controls to test the effectiveness of digestion and ligation. We ran digestion reactions for 15 minutes, 1 hour, 2 hours, 3 hours and 4 hours, and transformed all the products to test what the optimal time for a digestion reaction was. We then re-ligated the digestions and transformed them to test if the ligation reaction was working as expected. Our results showed that 15 minutes was enough time to carry out the restriction reactions, since there wasn’t a significant difference in number of colonies on the plate after transformations. However, there were several colonies present on the plate, albeit less than the number of colonies we ran for our control, which was simply undigested plasmid. This showed us that our restriction reaction wasn’t as efficient as expected. Our re-ligated products had more colonies than our restriction, but still significantly less than the control, being less than half the number of colonies of the control. In addition, a significant number of these colonies appeared to be undigested backbone based on our restriction controls.
We weren’t able to combine our plasmid via restriction cloning due to the ineffectiveness of the ligation and restriction reactions and a lack of budget to order new enzymes to test. Due to this, we decided to try a new method to combine our parts, Gibson cloning.
Gibson Assembly
This method requires the parts to have partially complementary sequences (around 20 bp), and a linearized plasmid to insert the parts into. The Gibson Master Mix then combines all the parts in the same reaction within 15 minutes. We ordered new parts so that our genes would have 20 bp complementarity on each side with its neighboring sequences. We also ordered new primers to linearize the plasmid. We chose to use the plasmid containing pLac and an RBS from the distribution kit to carry out our reaction. We designed our construct to look like the following:
| Part Type | Location | Function | Length |
|---|---|---|---|
| pLac + RBS | In backbone | Promoter for glpB | 220 bp |
| glpB | In gblock | Lyase: glyphosate tolerance | 930 bp |
| pTet + RBS | In gblock | Promoter for glpA | 69 bp |
| glpA | In gblock | Hph: glyphosate degradation | 1263 bp |
Our Gibson reaction was successful and yielded multiple colonies. To test whether it was our construct and not contamination, we digested our plasmid and visualized it on an agarose gel. The bands matched up to the lengths we expected to see. We then sequenced the construct to ensure that it was what we expected, and confirmed that the Gibson reaction yielded the construct shown above.
Project Testing
To test the efficacy of our construct we planned to carry out 2 phases of experiments. The first was to test if our construct, Rounddown, was more survivable in glyphosate as well as Roundup (the most common commercial formulation using that uses glyphosate). Running growth curves with Rounddown and a control with different concentrations of glyphosate and Roundup in LB would allow us to test this. Our second phase would involve growing Rounddown vs a control in glyphosate in LB and verifying that it does break down glyphosate. The varying concentrations could be tested through various methods such as NMR.
Herrera-Estrella, L, et al. "Cloning and Sequencing of the Genes Involved in Glyphosate Utilization by Pseudomonas Pseudomallei."Applied and Environmental Microbiology, American Society for Microbiology, 1 Feb. 1995, aem.asm.org/content/61/2/538.long?fbclid=IwAR3o5qOLyg0pj91y_k8B7KEcRnlVAt11NRG5kry9VUwBVzPBtK3FVaFeeiY.
