- Parts Overview -

Plasmid

We used several different backbones to clone our various inserts:

pUMP001

The pTE1877 backbone was kindly provided to us by the Tobias Erb group. It is a vector that can be shuttled through E. coli for use in Mex and is multicopy with an IPTG inducible promoter. It contains mCherry which is excised during cloning so that it functions as a disruptable marker. It has a kanamycin resistance gene so also indicates successful transformation when plated on media with kanamycin.

The assembled plasmid contains lutH under inducible expression of the P_L/O4/A1 promoter. LutH is a lanthanide transporter so increased transcription increases the uptake of lanthanides which maximises lanthanide scavenging [1].

pLNM001

The pMW224 backbone was kindly provided by the Anke Becker group. It is an empty vector that can be shuttled through E. coli for use in Mex. It has a tetracycline resistance gene so enables the selection of transformed cells once plated on media with tetracycline.

As we are engineering Mex to uptake more lanthanides than wild-type Mex by increasing lutH transcription, we are inducibly upregulating a cytosol-localised variant of the lanthanide binding protein lanmodulin (LanM) to mitigate stress. The lanM gene codes for lanmodulin which sequesters excess cellular lanthanides which reduces the stress that they cause. We also had to clone in lacIQ and a lac promoter (P_L/O4/A1) to control expression of lanM using IPTG [2].

LanM also acts as a buffer for free cellular neodymium, keeping it sequestered and unable to promote tar expression (see below) until it has accumulated sufficiently to saturate LanM. This is expected to optimise the bioaccumulation strategy.

pTAR001

The pMW220 backbone was kindly provided by the Anke Becker group. It is an empty vector that can be shuttled through E. coli for use in Mex. It has a gentamicin resistance gene so enables the selection of transformed cells once plated on media with gentamicin.

The insert, tar, encodes for an MCP. An MCP is a ligand sensing protein that promotes chemotaxis towards the attractant ligand [3]. We are using an aspartate MCP derived from E. coli under neodymium sensing mechanisms which are derived from Mex [4]. This will give Mex a foreign chemotaxis ability towards aspartate, a cheap and accessible chemoattractant, which is expressed in the presence of lanthanides. This means that once lanthanides have been accumulated, Mex then swims towards aspartate where the accumulated lanthanides can then be extracted. The insert is under a neodymium upregulated promoter, P_xoxF1[5].

pXOX001

The pET-28a(+) backbone was provided by the Alberti Lab, and can be bought from SnapGene. It is a bacterial vector for expression of N-terminally 6xHis-tagged proteins with a thrombin cleavage site [6]. Expression is induced by T7 RNA polymerase provided by the expression system E. coli BL21.

The XoxF insert is a PQQ-dependent periplasmic alcohol dehydrogenase [7]. We used this protein for our neodymium uptake assay to quantify how much neodymium was taken up using spectrophotometry, as outlined in the protocols.

XoxF forms inclusion bodies with this expression strategy, and requires resolubilisation and refolding to regain activity. We recommend an alternative expression strategy, such as that in a paper by Huang et al. (2018) [8].

We thank IDT for providing the gene block fragments and to Full Circle Labs for sequencing our plasmids.

Promoters

Vu et al. (2016), characterise the robust Ln-dependent upregulation of expression of mVenus downstream of the intergenic region prior to XoxF1. We use this intergenic region, which we label P_xoxF1, as a promoter for tar. As shown in figure 5A of the paper, expression from this promoter, here labelled xox1, is upregulated in the presence of lanthanides [5].

For inducible promoters, we assembled the lutH and lanM inserts under the IPTG inducible promoter P_L/O4/A1, selected due to its high maximum strength - shown in figure 6 relative to the metabolic enzyme P_mxaF promoter. We found that even leaky expression under this promoter was lethal to cells expressing lutH, and subsequent cloning strategies would use a weaker promoter.

Organisms

We used NEB Turbo Competent E. coli to transform our plasmid and generate stocks of our engineered bacteria. This is due to their high efficiency and fast colony growth.

For the XoxF protein expression, after purifying the plasmids through miniprep from the Turbo cells, we transformed into E. coli BL21 Competent cells due to their high levels of recombinant protein expression and easy induction.

For the other plasmids, after miniprep of the Turbo cell plasmids, the cells were transformed into M. extorquens AM1 for characterisation. Mex was used as it contains the natural lanthanide-binding protein lanmodulin and already contains lanthanide uptake and sensing machinery [9]. The AM1 strain was used specifically as it is the most well characterised.