Part:BBa_K3802000
PgsA-MlrA fusion protein
Contents
Sequence and Features
This fusion protein aims to anchor MlrA, or microcystinase, an enzyme capable of degrading freshwater toxin microcystin-LR (MC-LR) to the outer membrane of E. coli . This part puts together a codon-optimized subunit of PgsA (poly-γ-glutamic acid synthetase) (BBa_K2963020), an anchoring motif to MlrA with 6x His (BBa_K1907002), with a 2 amino acid Gly-Ser linker (BBa_J18920). The addition of the His tag to MlrA gives this part the ability to be purified using Nickel affinity chromatography and detected using Western blotting with anti-His antibodies. This BioBrick was put under the control of an IPTG inducible ptac promoter (BBa_K3802001), as using a constitutive promoter could potentially lead to the depletion of resources for the bacteria.
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1792
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 827
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 813
Illegal NgoMIV site found at 2323 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 304
Illegal BsaI.rc site found at 2160
Usage and Biology
Our project aims to develop a novel MC-LR degradation solution by utilizing recombinant E. coli that heterologously over-express MlrA, an enzyme known to degrade MC-LR in the native Sphingomonas sp [1]. This composite part aimed to improve upon previous systems of MC-LR detection by creating a method where this toxin could be degraded in the outer membrane of E.coli . The advantage of this system is that MC-LR does not have to be transported into the cytosol of E. coli for degradation, but rather can be directly broken down if MC-LR is within proximity of E. coli.
This fusion protein links MlrA with the gene for PgsA (poly-γ-glutamate synthetase A), a protein natively found in Bacillus subtilis [2] Though past iGEM teams such as JNU- China from 2019 have genetically engineered Corynebacterium glutamicium to produce PgsA for various applications such as food additives, anti-freeze, protective antigen coupling, PgsA can also act as an anchoring motif. This protein has been used to successfully and heterologously express enzymes and proteins such as α-amylase, lipase B, Laccase COTA, and VP2--an antigen for IBDV virus in chickens [5][3][4].
PP1 Assay
In order to indirectly test for MlrA expression, PP1 assay was used to quantify how much microcystin remained after incubating transformed E. coli with MC-LR. However, several issues arose when attempting to replicate the results from [6]. Firstly, the standard curve generated showed no inhibition by MC-LR, regardless of the concentration used. On the other hand, the curve from the literature demonstrated a clear decrease in relative PP1 activity with increasing MC-LR concentration. To determine whether lack of inhibition was due to the MC-LR solution used or a procedural error, it was decided to test another inhibitor of PP1. Given the limited time frame, it wasn’t possible to order a well-characterized, selective PP1 inhibitor, such as okadaic acid or tautomycin. Instead, 25mM dibasic sodium phosphate was used, since it is an inhibitor of other phosphatases and was readily available [7]. However, unexpectedly, results from the PP1 assay demonstrated activation, not inhibition, in the presence of sodium phosphate (Figure 1). This is indicated by the fact that the log of the relative PP1 activity is positive and increases with increasing concentrations of sodium phosphate (as a percent of total reaction volume)—an inhibitor would produce negative values that increase in magnitude with greater inhibitor concentration.
Figure 1. Relative PP1 activity rate, expressed as log (exposed PP1 activity rate/control PP1 activity rate), for varying concentrations of sodium phosphate.
Testing the Anchoring Construct
Despite the mixed results when generating the calibration curve, samples of transformed and wild-type bacteria were tested by the same protocol. Both cell lysate and whole cells were tested in order to get a sense for whether MlrA was present primarily on the outer membrane, as was hoped, or the cytoplasm, as in previous work [1].However, as seen in Figure 2, these results proved to be inconclusive. While cell lysate of transformed BL21 did seem to result in reduced PP1 activity relative to the negative control (without sample exposure) at some concentrations, this was not true for all concentrations. Furthermore, these points did not show a strong correlation (R2=0.139). Similarly, data for whole cell samples from control and transformed E. coli had R2 values of 0.065 and 0.111, respectively. Given both the unsuccessful attempts at creating the calibration curve and the absence of strong trends, it is unlikely that these results are an accurate representation of the MlrA expression levels.
Figure 2. Relative PP1 activity rate, expressed as log (exposed PP1 activity rate/control PP1 activity rate), for varying concentrations of sample.
References
[1] 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
[2] Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M.-H., Fukuda, H., & Kondo, A. (2005). Display of active enzymes on the cell surface of Escherichia COLI using PgsA ANCHOR protein and their application to bioconversion. Applied Microbiology and Biotechnology, 70(5), 564–572. https://doi.org/10.1007/s00253-005-0111-x
[3] Zhang, Y., Dong, W., Lv, Z., Liu, J., Zhang, W., Zhou, J., Xin, F., Ma, J., & Jiang, M. (2018). Surface display of BACTERIAL Laccase COTA on Escherichia COLI cells and its application in Industrial Dye Decolorization. Molecular Biotechnology, 60(9), 681–689. https://doi.org/10.1007/s12033-018-0103-6
[4] Maqsood, I., Shi, W., Wang, L., Wang, X., Han, B., Zhao, H., Nadeem, A. M., Moshin, B. S., Saima, K., Jamal, S. S., Din, M. F., Xu, Y., Tang, L., & Li, Y. (2018). Immunogenicity and Protective efficacy of orally administered RECOMBINANTLACTOBACILLUS Plantarumexpressing VP2 protein against IBDV in chicken. Journal of Applied Microbiology, 125(6), 1670–1681. https://doi.org/10.1111/jam.14073
[5] MASEDA, HIDEAKI., SHIMIZU, KAZUYA, DOI, YOSHIAKI, INAMORI, YUHEI, UTSUMI, MOTOO, SUGIURA, NORIO, & KOBAYASHI, MICHIHIKO (2012). MlrA located in the inner membrane is essential for Initial degradation Of microcystin In sphingopyxis sp. C－1. Japanese Journal of Water Treatment Biology, 48(3), 99–107. https://doi.org/10.2521/jswtb.48.99
[6] 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
[7]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
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