Team:Stony Brook/Engineering

iGEM SBU 2021

Navigating to Engineering

Engineering Success

Anchoring Construct

For the PgsA-MlrA construct, a plasmid was designed to express MlrA on the membrane surface of E. coli. A DNA insert containing the anchoring and enzyme genes was attached to a DNA backbone via HiFi assembly to form the final plasmid. Firstly, this insert contains a ptac promoter, which is induced by IPTG (Browning et al., 2017). An inducible promoter was used because a constitutive promoter could run the risk of killing the bacteria by depleting its resources. To ensure protein expression, an Anderson family ribosome binding site (BBa_J61100) was used because it is suitable for general protein expression in E. coli. Next, the DNA insert contains the PgsA (anchoring motif) gene connected to the MlrA (microcystinase) gene with a 2 amino acid Gly-Ser linker. PgsA (BBa_K2963020), the GS linker (BBa_J18920), and MlrA (BBa_K1907002) were parts from the registry used to form a new composite part. To ensure these genes can be expressed in E. coli, this composite part was codon optimized via IDT’s codon optimization tool. The terminator.. Overhangs were built into the PgsA-MlrA gBlock to avoid an additional PCR step that could potentially introduce errors in the sequence.

Figure 1. Final DNA insert for PgsA-MlrA.

For the backbone or vector pKSI-1 was used because of its efficiency in bacterial expression and high copy number. This vector has ampicillin resistance.

For the E. coli chassis, it was decided to use BL21(DE3). BL21(DE3) is commonly used for recombinant protein expression because it contains a T7 RNA polymerase gene under the control of a lacUV5 promoter in the bacterial genome, which allows for high protein expression (Rosano et al., 2019).

Figure 2. Final plasmid for the Anchoring Construct.

TatExpress: Periplasmic Secretion

The design of the TatExpress construct was based on the paper published by Browning et al., “Escherichia coli “TatExpress” strains super‐secrete human growth hormone into the bacterial periplasm by the Tat pathway”. As mentioned on the contribution page of the wiki, heterologous expression of MlrA typically results in the protein being localized to the cytoplasm ((Dziga et al., 2012)). Given that microcystin is found extracellularly and does not freely diffuse into the cytoplasm, this results in limited exposure of MC-LR to MlrA and, ultimately, inefficient degradation. In creating the TatExpress strain described by Browning et al., this issue could be addressed as machinery to export MlrA would be upregulated.

The promoter chosen for the insert was pTet (BBa_K3171173), as it can be used as both an inducible and constitutive promoter. The insert also included a TorA signal sequence and mature four amino acid TorA protein. These sequences were obtained from Browning’s 2017 paper, but a similar part can be found in the registry (BBa_K1012002). This signal peptide, fused to the N-terminus of MlrA, would allow MlrA to be transported via the Tat machinery. Finally, the RBS and terminator used were BBa_J61100 and BBa_B0010, respectively.

Figure 3. Final plasmid for the TatExpress construct.

Detection System

The detection portion of the project was based on the bacterial two-hybrid system and required three inserts: one for each half of the adenylyl cyclase catalytic domain, and one encoding for green fluorescent protein (GFP), which would be expressed in the presence of microcystin.

The first insert codes for the catalytic domain of protein phosphatase 1 (PP1) and one fragment of the adenylyl cyclase catalytic domain, T18. A strong constitutive promoter from the Anderson family was used (BBa_J23112), followed by an Anderson family RBS (BBa_J61102), the gene for PP1 (BBa_K1012001), a 54 amino acid Gly-Gly-Ser linker (BBa_K3128010), and the gene for T18 (BBa_K1638004). Finally, an rrnBT1-T7Te terminator (BBa_B0015) was used.

Figure 4. First insert contains the genes for PP1 and T18.

The second insert codes for glutathione (GSH) and the other fragment of the adenylyl cyclase catalytic domain, T25. The parts used were a constitutive promoter (BBa_J23112), RBS (BBa_J61102), GSH (BBa_K2571001), T25 (BBa_K1638002), and rnpB-T1 terminator (BBa_J61048) were used.

Figure 5. Second Insert contains the genes for GSH and T25.

The last insert codes for green fluorescent protein (GFP) and is responsible for the output of the detection system. A cAMP inducible promoter was used (BBa_K121011), followed by an RBS (BBa_J61102), the gene for GFP (BBa_E0040), and an rrnB T1 terminator (BBa_B0010).

Figure 6. Last Insert contains the gene for green flurescent protien (GFP.

Initially during the design process, all three inserts had the same terminator. However, it was decided that the three terminators should be varied to prevent off-target annealing of the overhangs during HiFi assembly.

The vector used for this construct was pUC19.

Figure 7. Final plasmid for the Detection system.


Browning, D. F., Richards, K. L., Peswani, A. R., Roobol, J., Busby, S. J. W., & Robinson, C. (2017). Escherichia coli “TatExpress” strains super-secrete human growth hormone into the bacterial periplasm by the Tat pathway. Biotechnology and Bioengineering, 114(12), 2828–2836.

Dean, R. L. (2002). Kinetic studies with alkaline phosphatase in the presence and absence of inhibitors and divalent cations. Biochemistry and Molecular Biology Education, 30(6), 401–407.

Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation of microcystinase. Toxicon, 59(5), 578–586.

Moore, C., Juan, J., Lin, Y., Gaskill, C., & Puschner, B. (2016). Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases. Marine Drugs, 14(3), 54.

Rosano, G. L., Morales, E. S., & Ceccarelli, E. A. (2019). New tools for recombinant protein production in Escherichia coli : A 5‐year update. Protein Science, 28(8), 1412–1422.