Aims and Objectives
We are working to develop a strain of bacteria that scavenges for lanthanide ions and via an engineered foreign chemotactic system, swims towards aspartate (chemoattractant).
Our project has three different aspects to it: uptake, sequestration and chemotaxis. For increased uptake, transcription of the lanthanide transporter, lutH, would be increased [1]. To minimise stress caused by this increased uptake, a protein, lanmodulin, would be upregulated which binds to uptaken lanthanides, sequestering them and therefore reducing stress [2]. An MCP would be engineered which is a transmembrane receptor which senses attractant compounds, promoting chemotaxis [3]. This would be under a Nd-upregulated promotor, XoxF, meaning that when the bacterium has accumulated the lanthanides, it would swim towards a collection point (the chemoattractant) so lanthanides can be extracted.
Adjustments and considerations had to be made regarding project design throughout.
Chassis
We use Methylobacterium extorquens (Mex) as our chassis due to the fact it already contains lanthanide uptake and sensing machinery. It also contains the natural lanthanide binding protein lanmodulin (LanM) [2]. This bacterium poses many experimental challenges, not least that it is only partially characterised; and that it has a slow doubling time of between 3 and 4 hours [4]. However, the existence of lanthanide handling pathways was a very attractive starting point for the iGEM timeframe.
![Structure of lanmodulin from [5]](https://static.igem.wiki/teams/5277/images/design2.webp)
Choosing a chemoattractant
We planned to engineer foreign MCPs into Mex derived from Escherichia coli. However, the MCPs for Mex are not well characterised, so we needed to determine what Mex is already attracted to. To do this, we completed swim plate assays with a similar set up to the swim plates of the proof of concept experiment using hard agar plugs made from various known E.coli chemoattractants: aspartate, serine, maltose and galactose [6][7]. These didn’t show significant results as the bacteria weren’t motile so we performed a directed evolution experiment using swim plates to generate more highly motile bacteria.
We then did swim plates, as detailed on the protocols page, to try to find an E. coli chemoattractant that Mex wasn’t attracted to. However, these showed no significant results. Due to the timespan of the project, the gene block fragments for the MCP had to be ordered so we were unable to continue testing the existing Mex chemotaxis and instead opted to use an aspartate MCP derived from E. coli. If Mex was attracted to aspartate, the system would still have been functional but less efficient so in the interest of time we decided to order the DNA.
Plasmid Construction
We began cloning of lutH, lanM, tar (aspartate MCP) and xoxF to their respective plasmids, as detailed in the parts overview. We opted for Gibson Assembly for our cloning as our plasmids weren’t compatible with Golden Gate. Suitable care was taken to ensure plasmids were genetically manipulable after transformation by (re)introducing restriction sites at key positions into the assembled plasmids.
- pTE1877 - lutH (pUMP001)
XbaI and HindIII were used for the restriction digest of pTE1877. This excises mCherry so that when lutH is inserted, there is a visual indicator as to whether the transformation is successful - mCherry is a disruptable marker. - pMW224 - lanM (pLNM001)
ScaI was used for the restriction digest of pMW224. - pMW220 - tar (pTAR001)
ScaI was used for the restriction digest of pMW220. - pET28a(+) - xoxF (pXOX001)
BamHI was used for the restriction digest of pET28a(+).
Results of the transformations can be seen in the results section.
Design of our neodymium concentration assay
We designed a spectrophotometric assay to quantify neodymium concentrations due to the costs and complexity associated with ICP-MS. The theory behind the assay is detailed comprehensively in the contributions section, and involves manipulating Michaelis Menten kinetics. The theory is for generally quantifying chemicals which are used by enzymes as cofactors and thus is unsurprisingly adaptable for metal ion quantification. Our specific assay uses the lanthanide-dependent methanol dehydrogenase XoxF.
XoxF oxidises methanol to formaldehyde using the redox cofactor PQQ, which is reduced [8][9]. Phenazine methosulfate (PMS) can reoxidise PQQ, becoming reduced and reduced PMS can reduce DCPIP. DCPIP is blue when oxidised and colourless when reduced - a change that can be measured spectrophotometrically at 600nm (DCPIPs peak absorbance). Figure 3 details the chemistry visually. Care must be taken when conducting the assay as it uses the light-sensitive chemical PMS, so dark conditions had to be maintained throughout.
Proof of concept design
The design for the proof of concept is similar to that of the experiments used for choosing the chemoattractants. We used the swim plate which had the Mex mixed in with the soft agar and an aspartate plug in the middle. Specific components are detailed on the protocols page. The set up was adapted from the unsuccessful chemoattractant experiment. For example, we reduced the percentage w/v of the soft agar from 0.3% to 0.2% so the bacteria could move more easily. Additionally, we waited for the agar mix to be cool but not set before adding the Mex, in case the high temperature killed them.
References
^[1] Shiina W, Ito H, Kamachi T. (2023) Identification of a TonB-Dependent Receptor Involved in Lanthanide Switch by the Characterization of Laboratory-Adapted Methylosinus trichosporium OB3b. Appl Environ Microbiol. 89(1)
^[2] Mattocks, J.A., Jung, J.J., Lin, CY. et al. (2023) Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer. Nature 618, 87–93
^[3] Parkinson J.S., Ames P., Studdert C.A.(2005). Collaborative signaling by bacterial chemoceptors. Current Opinion in Microbiology. 8(2).116-121.
^[4] Strovas TJ, Sauter LM, Guo X, Lidstrom ME. (2007) Cell-to-cell heterogeneity in growth rate and gene expression in Methylobacterium extorquens AM1. J Bacteriol. 189(19):7127-33.
^[5] Park J, Cleary MB, Li D, Mattocks JA, Xu J, Wang H, Mukhopadhyay S, Gale EM, Cotruvo JA Jr. (2022) A genetically encoded fluorescent sensor for manganese(II), engineered from lanmodulin. Proc Natl Acad Sci U S A. 119(51)
^[6] Matilla M.A., Gavira J.A., Krell T (2023) Accessing nutrients as the primary benefit arising from chemotaxis. Current Opinion in Microbiology. 75.
^[7] Adler J, Hazelbauer GL, Dahl MM. (1973) Chemotaxis toward sugars in Escherichia coli. J Bacteriol. 115(3):824-47.
^[8] Anthony C, Zatman LJ. (1964)The microbial oxidation of methanol. 2. The methanol-oxidizing enzyme of Pseudomonas sp. M 27. Biochem J. 92(3):614-21.
^[9] Huang J, Yu Z, Chistoserdova L(2018) Lanthanide-Dependent Methanol Dehydrogenases of XoxF4 and XoxF5 Clades Are Differentially Distributed Among Methylotrophic Bacteria and They Reveal Different Biochemical Properties. Front Microbiol.9:1366.