Team:Stony Brook/Engineering
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.
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).
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.
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.
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.
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).
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.
References
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. https://doi.org/10.1002/bit.26434
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. https://doi.org/10.1002/bmb.2002.494030060138
Dziga, D., Wladyka, B., Zielińska, G., Meriluoto, J., & Wasylewski, M. (2012). Heterologous expression and characterisation of microcystinase. Toxicon, 59(5), 578–586. https://doi.org/10.1016/j.toxicon.2012.01.001
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. https://doi.org/10.3390/md14030054
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. https://doi.org/10.1002/pro.3668
Anchoring Construct
The pKSI-1 vector was ordered from Addgene as an agar stab and was replated using the quadrant method. After overnight culture, colonies were inoculated in liquid Luria-Bertani (LB) media. After another overnight culture, we miniprepped using the Monarch Plasmid Miniprep kit to isolate the vector DNA for use in HiFi assembly.
For HiFi assembly, the vector was linearized by restriction enzyme digestion using the enzymes BamHI and HindIII. The NEBuilder HiFi DNA Assembly kit was used to ligate the DNA insert PgsA-MlrA with the vector, pKSI-1.
The plasmid was transformed into competent DH5-alpha E. coli, chosen because it can produce a high copy number of plasmids. NEB’s protocols for chemical transformation via heat shock were followed. Bacteria containing the plasmid were plated on agar plates with ampicillin. Once the colonies grew, some cultures were placed in liquid LB media and incubated overnight. Next, the cultures were miniprepped to isolate the final plasmid and nanodropped to ensure high DNA concentration.
Before transforming the final plasmid into BL21, it was important to ensure that the assembly step had proceeded as expected. Thus, a restriction enzyme digest was done using the enzymes SpeI and Kpn1. Since two Kpn1 restriction sites and one SpeI restriction site were present in the plasmid, three bands should appear on a gel. The size of the bands corresponded to the simulated agarose gel produced by SnapGene.
The final plasmid was chemically transformed using heat shock in competent BL21(DE3) and cultured on agar plates with ampicillin resistance.
Testing the Anchoring Construct
An assay for MC-LR degradation was used to indirectly test for the presence of functioning MlrA protein. For this assay, the protocols adopted were based on work by Moore et al. and the 2014 Peking iGEM team, who also expressed MlrA and tested MC-LR degradation. This assay relies on the premise that MC-LR is an inhibitor of protein phosphatase 1 (PP1). PP1 catalyzes the conversion of p-nitrophenyl phosphate (pNPP) to p-nitrophenol (pNP), producing a yellow color. By monitoring absorbance at 405 nm in the presence of pNPP and PP1, the reaction rate, and indirectly the concentration of MC-LR, can be measured. High MC-LR concentration leads to greater PP1 inhibition and a less yellow solution (low absorbance). On the other hand, a low MC-LR concentration would be associated with less PP1 inhibition, faster conversion of pNPP to pNP and higher absorbance. If MlrA is correctly localized to the outer membrane, this would lead to lower MC-LR concentration, less PP1 inhibition and, in turn, increased absorbance.
A calibration curve was generated to determine the relative PP1 activity rate at MC-LR concentrations between 0 μg/L and 1 μg/L. At higher MC-LR concentration, the relative PP1 activity rate should decrease, as seen in the curve produced by Moore et al. However, in our attempts to generate this curve, the PP1 activity rate remained unchanged despite increasing microcystin concentration.
In order to indirectly test for MlrA expression, we hoped to use the PP1 assay to quantify how much microcystin remained after incubating transformed E. coli with MC-LR. We first incubated whole cells and cell lysate from our transformed BL21(DE3), as well as wild-type E. coli, with MC-LR at a final concentration of 100ng/L at 25 °C in accordance with the 2014 Peking iGEM team’s protocol. After 24 hours, we terminated the reaction by heating all solutions to 98 °C. Subsequently, we used the protocol from Moore et al. for the PP1 assay.
Learning experience
Throughout this design cycle, there were some shortcomings that prevented the team from achieving ideal results. First, the transformed BL21(DE3) should have been tested for protein expression. Though RE digest and gel electrophoresis were conducted to check if the final plasmid contained the expected size insert, there was no guarantee that the E. coli would express MlrA, or that it would be anchored to the membrane. Our advisor, Dr. Gergen and teaching assistant Andrew Sillato, agreed that a western blot should have been performed to confirm PgsA-MlrA protein expression. However, this was not possible due to time constraints.
Also another issue our team faced was conducting the assay. As mentioned before, it was found that increasing microcystin concentration had no effect on PP1 activity, indicating an inactive inhibitor. In order to confirm this, it was decided to test our protocol with a different PP1 inhibitor. Therefore, we used sodium phosphate dibasic at varying concentrations in place of MC-LR, as inorganic phosphate is known to be an inhibitor of protein phosphatases (Dean, 2002).
