Team:Lambert GA/Design
DESIGN
ORIGINAL HKUST-RICE 2015 IGEM DESIGN
After months of research, Lambert iGEM’s NarL system is designed using HKUST-Rice 2015’s part BBa_K1682018. Lambert iGEM’s NarL system is modified to avoid the original design’s terminators due to problematic DNA synthesis in IDT (Integrated DNA Technologies). The promoter BBa_J23106, an Anderson Promoter, constitutively produces TetR, which represses the PtetO promoter that produces NarL. In the presence of nitrate, NarX will phosphorylate NarL, activating it; however, if no nitrate is present, NarX will not phosphorylate NarL. To analyze data, Lambert iGEM came across two promoters that could produce the downstream reporter green fluorescent protein (GFP): PyeaR and PdcuS. Both promoters are repressed by phosphorylated NarL; however, PdcuS is slightly more sensitive than PyeaR and is not affected by NarP (See Nar Engineering Success: Background Research).
NarL CONSTRUCT DESIGN
Lambert iGEM initially designed its SnapGene constructs for Gibson Assembly as shown in Figure 3.1. However, the team later switched to restriction cloning (Figure 3.2) to optimize the success rate since IDT discouraged running PCRs on parts larger than 1kb. Nevertheless, both the Gibson Assembled and Restriction Cloning construct consist of the same basic parts: PtetO promoter with tet operators, NarL gene, PdcuS, GFP, tetR, and the backbone, pSB1C3 (a standard high copy backbone). The Gibson Assembled Construct also contains specifically designed primers for insert and vector PCR.
Figure 1. The figure above shows the final NarL Gibson Assembled Construct.
Figure 2. The figure above shows the final NarL Restriction Cloning Construct.
NarP CONSTRUCT DESIGN
Lambert iGEM also designed its novel NarP insert BBa_K3411020. Similar to the NarL composite part, the promoter BBa_J23106, a constitutive Anderson Promoter, produces TetR, which represses the PtetO promoter, producing NarP. In the presence of nitrite and/or nitrate, NarQ will phosphorylate NarP, activating it, but if there is no nitrite and/or nitrate present, NarQ will not phosphorylate NarP. In the presence of phosphorylated NarP, a promoter is activated, triggering GFP expression. Lambert iGEM came across two potential promoters: nirB and nrfA. Both promoters produce GFP in the presence of phosphorylated NarP; however, data derived from nirB induced GFP expression will be easier to interpret (See Project: Nar Engineering Success). Therefore, Lambert iGEM’s final Gibson Assembled NarP Construct (Figure 3.3) consists of five basic parts: the NarP gene, the Anderson promoter BBa_J23106, sGFP (superfolder GFP), TetR, PtetO, and nirB. Since the final NarP insert is 2991 base pairs, restriction cloning would not be efficient for assembling the biosensor, so the team did not create a restriction cloning construct for NarP.
Figure 3. The figure above shows the final NarP Gibson Assembled Construct.
NarL UPDATED CONSTRUCT
After further research and cloning, Lambert iGEM redesigned the NarL Insert Construct for Gibson Assembly by separating the construct into two parts: 1) PtetO, NarL gene, PdcuS promoter, and the first half of superfolder GFP and 2) the second half of sGFP, BBa_J23106, and TetR. The team plans on assembling the two inserts and backbone using Gibson Assembly; by splitting the construct into two fragments, each fragment will be around 1372 bp, which is less than the successfully amplified Pho insert (See Wetlab: Pho) .This construct has all the same parts as the initial NarL construct shown in Figure 3.1.
Figure 4. The figure above shows the final two-fragment and one vector Gibson Assembled Construct built in Snapgene.