iGEM 2024 — Team warwick

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{% block title %}Lab Book{% endblock %}
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Our notebook is available to download [here (PDF)](https://static.igem.wiki/teams/5277/images/notebook/output.pdf).

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## Introduction

---

Throughout our project's development, we have iterated through
each of the stages of the engineering cycle, in order to ensure
an efficient and robust engineering process. Here we describe
our usage of the design-build-test-learn cycle in the different
aspects of the project.

![Design Built Test Learn cycle](https://static.igem.wiki/teams/5277/images/engineering0.webp)

iGEM engineering cycle

## Taxis

---

### Design

Engineer in foreign
chemotaxis under Nd upregulation =>

1. WT must not natively show positive or negative
   migration
2. Easily integrable => Use system from gram negative
   (same as *M. extorquens*).

1. => Test *M. extorquens* native taxis to the
   below *E.coli* chemoattractants.
2. => *E. coli* MCPs (ligand sensing component of
   taxis), which are:
   * Tar†: Aspartate, Maltose\*
   * Tsr†: Serine
   * Tap: Ribose\*, Galactose\*
   * Trg: Dipeptides\*
   * Aer: ~~O2~~ Experimental
     setup too complicated
† Abundant in *E.coli*, \* bind MCP via periplasmic binding
proteins

---

### ~~Build~~

No build for WT characterisation

---

### Test

We were interested in chemotaxis, and therefore had
to decide on a motility assay and optimise
for *M. extorquens*. We chose a
qualititive assay that gives a robust binary result within
an hour of inoculation, and uses small plates such that it does
not waste resources.

![whiteboard photograph number 1](https://static.igem.wiki/teams/5277/images/engineering1.webp)
![whiteboard photograph number 2](https://static.igem.wiki/teams/5277/images/engineering2.webp)

Qualitative, Growth-Unsupportive Chemotaxis Assay

---

### Learn

Unfortunately, we got no positive results, even from positive
control. Therefore, we moved on to a protocol that i) can be
troubleshot more easily, and ii) better matches the setup for
our proof of concept.

---

### Design

The new design facilitates taxis over the course of the growth
timeframe, and also allows a quantitative measurement of the
cells’ propensity to move towards the chemoattractants.

---

### Test

![whiteboard photograph number 3](https://static.igem.wiki/teams/5277/images/engineering3.webp)

Quantitative, Growth-Supportive Chemotaxis Assay

---

### Learn

We see colonies mostly ascribing to the third option – no motility. This was highly concerning, as it suggested that the strain of *M. extorquens* we had had been domesticated such that it had lost motility. We knew we needed to somehow reintroduce it, but that using genetic engineering would be a largely blind process.

---

### Design

We opted to perform a series of successive swim plates, each time taking bacteria from the farthest point that it grew from inoculation, and reinoculating with that.

---

### Build

This directed evolution was repeated until we saw sufficient
motility to be characterisable. We grew up two motile colonies,
made glycerol stocks of them and named them ‘*Mex* mot A’;
and ‘*Mex* mot B’. We used *Mex* mot A for all future
experimentation and transformation.

### Test

We reattempted the assay in the previous cycle with our *Mex* mot A strain.

### Learn

We successfully observed negative results for the *E. coli* chemoattractants. This gave us a choice of chemoattractants, so we were able to pick one that is:

* Easy to engineer
* Cheap at industrial scale

### Design

We chose Tar for aspartate sensing, because:

* The MCP binds aspartate directly => only one component => simple.
* Abundant in *E. coli* => well characterised activity.
* Asp is a chemically simple amino acid => cheap and readily available.

For Nd regulation, we inserted the intergenic region upstream of *M. extorquens*' *xoxF1*, which is characterised as being robustly upregulated in lanthanides (La3+, Ce3+).

---

### Build

![whiteboard photograph number 4](https://static.igem.wiki/teams/5277/images/engineering4.webp)

Building Artificial Chemotaxis

---

### Test

Using aspatate in plug, perform Ex (2) from
previous cycle with:

1. *Mex* mot A
2. *Mex* mot A + P*xoxF1*-*tar*

---

### Learn

We saw some limited movement towards Asp in
the transformant
and **not** in the
untransformed *Mex* mot A strain
at 0.2% w/v agar.

=> Successful endowment of aspartate taxis in Nd-supplemented media.

## Bioaccumulation

---

We wanted to optimise the capacity of M. extorquens for
lanthanide uptake, so we designed a bioaccumulation strategy, as
below.
![whiteboard photograph number 5](https://static.igem.wiki/teams/5277/images/engineering5.webp)

Bioaccumulation Strategy

Therefore, we planned to engineer in LutH and LanM under inducible, titratable regulation, to test for optimal expression levels.

---

### Build

![whiteboard photograph number 6](https://static.igem.wiki/teams/5277/images/engineering6.webp)

Building Bioaccumulative Systems

With our promoter choice, even leaky expression of *lutH*
was observed to be lethal to cells.

---

### Test: Uptake

![whiteboard photograph number 7](https://static.igem.wiki/teams/5277/images/engineering7.webp)

Test: Uptake stage

1. Successive growths in Nd-supplemented media
   * Grow, then remove majority of cells by centrifugation, and save a sample of media.
   * Allow regrowth repeatedly until cells fail to grow.

   This assay measures the reduction in Nd concentration, which is in theory more sensitive to conditions where the majority of Nd is uptaken.
2. Overnight uptake
   * Grow to late exponential in absence of Nd, then induce overnight with Nd.
   * Centrifuge and wash cells repeatedly, collecting the cells for sampling after the last wash.

   This assay measures the uptake into cells directly, and allows standardisation by cell density. In theory it is more sensitive to conditions where cell uptake of Nd is limited.

Limited use of ICP-MS caused us to decide to run the assay 2) only.

---

### Learn

We found WT uptake was extremely efficient at the concentrations of Nd that we used. Therefore, we did not redesign the expression construct for weaker expression of LutH, because it was no longer considered an effective optimisation. This choice was supported by our [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) by this stage.

*lanM* did result in increased uptake per cell, which supports our idea of using it for neodymium sequestration. Nevertheless, it was not deemed necessary to optimise the design of *lanM* for uptake, because the WT was sufficiently efficient.

---

### Test: Stress

![whiteboard photograph number 8](https://static.igem.wiki/teams/5277/images/engineering8.webp)

Aim for stress reduction

1. Growth curves at variable [Nd]
   * High throughout
   * Highly quantified
   * Limited access to plate reader as undergraduate students
2. Viability plates
   * Magnitudes of growth observable
   * No specialist equipment

We decided to go with viability plates, because we expected our results to be spread across several magnitudes of neodymium concentrations.

![whiteboard photograph number 9](https://static.igem.wiki/teams/5277/images/engineering9.webp)

Viability Plate Setup

---

### Learn

Inconsistent results across similar experiments cast doubt on results, but initial analysis is promising. More repeats are required for a robust claim of success, but LanM does appear to reduce stress manyfold, in line with predictions from our [model](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

## Protein Purification

---

### Design

An enzymatic assay for [Nd] quantification. Details of assay chemistry on [Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) / [Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D) pages.

Here, we focus on XoxF purification.

XoxF = approx. 65 kDa, 4 Cys-S-S-Cys, periplasmic, dimerizes

Initial expression system: *E. coli* BL21, pET-28a(+)

---

### Build

![whiteboard photograph number 10](https://static.igem.wiki/teams/5277/images/engineering10.webp)

Building XoxF Expression System

### Test

Purify with Ni2+ column

1. Assay
2. SDS-PAGE

---

### Learn

Assay failed to show any activity.
SDS-PAGE showed protein was insoluble. We determined that we needed to change conditions to promote solubility.

---

### Design

Retry using cell lysate directly, supported by literature.

---

### Build

Same system

---

### Test

No activity

---

### Learn

Protein is insoluble, at least by lysis.

---

### Design

Retry with 4-fold reduced induction & in vivo assay

---

### Build

Reduce [IPTG] from 1 mM to 0.25 mM

---

### Test

Tested both, no activity

---

### Learn

Expression system is poor; pET-28a(+) does not facilitate the biophysical properties of XoxF

---

### Design

Resolubilise and refold protein

---

### Build

Extract inclusion bodies, resolubilise and refold them. Limitations with available lab equipment reduced the viability of the refolding step, as we had to perform dilution instead of dialysis.

---

### Test

Protein resolubilisation was succesful, but no activity with refolded protein

---

### Learn

In hindsight, had we had more experience with protein work across the team, we would have recognised that the expression system we used was inappropriate for a protein with the biophysical characteristics of XoxF, and we would have performed an existing protocol in the literature for purification

---

### Design

Transition to an *M. extorquens*-based expression system, as detailed as giving soluble protein in the literature

---

### Build

Limited by time constraints of iGEM season

## Proof of Concept

---

### Design

The bacterial strains used are those generated in previous cycles, but the setup still requires design. A particular issue was the collection of samples, because the soft agar cannot be excised effectively – the agar reforms around a blade.

![whiteboard photograph number 11](https://static.igem.wiki/teams/5277/images/engineering11.webp)
![whiteboard photograph number 12](https://static.igem.wiki/teams/5277/images/engineering12.webp)

Proof of Concept Experimental Setup

---

### Build

In order to collect samples reliably, we designed a simple
3D-printed ‘cookie cutter’, detailed on the [protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocols%27%29%20%7D%7D) page. The
cutter is stabbed into the plate with gridlines pre-drawn, and the
soft agar is pipetted up.

This produces **robust, reliable, reproducible results.**

The samples can then be solubilised with a chaotrope, and sonicated to lyse. Storage at -20 °C is good to prevent cell regrowth.

---

### Test

Run assay, collect samples. Samples quantified by:

1. ICP-MS
   * Gold standard
   * Expensive per sample
2. XoxF assay
   * Cheap per sample
   * Quick
   * Benchtop

---

### Learn

We saw promising [results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)!

We analysed different strains/conditions to assess the impact of our genetic components.
See [results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) for discussion.

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## Overview

---

*In vivo* projects are invariably complicated by the many
moving parts in biological systems. It is therefore an important
goal in synthetic biology to modularise genetic components and
their individual functions, so that different functional parts can
be combined in any number of ways without significantly
compromising the integrity of individual components.

BEACON’s project was composed of several modular aspects, each
serving independent purposes. It is intended that, with the
exception of the complete proof of concept, any of the following
sections can be read, understood and used without the need for
confusing crosstalk.

## Cloning

## Transformation of *lanM* into *Methylobacterium extorquens* (*Mex*)

---

Gibson assembly of the *lanM* gene into pMW224 was
successful. We successfully transformed
the *lanM*-pMW224 plasmid into *E.coli*, as
evident by the positive colony PCR (Figure 1), restriction digest
and sequencing results (Figure 2).

![Colony PCR of the lanM + PMW224 plasmid in E.coli. Plasmid was inserted via heat shock into DH5alpha competent E.coli on LB+tetracycline plates.](https://static.igem.wiki/teams/5277/images/results1.webp)

**Figure 1 - Colony PCR of the pLNM001
plasmid in *E.coli*.** Plasmid was inserted
via heat shock into DH5alpha competent *E.coli*
DH5α on LB+tetracycline plates.The expected length
of the PCR product was 641bp.

![Sequencing result of pLNM001 compared to the expected result](https://static.igem.wiki/teams/5277/images/results2.webp)

**Figure 2 - Sequencing result of pLNM001
(bottom) compared to the expected result
(top).**

The alignment is not 100% identity, but each individual
mismatch was assessed for impact on function, and it was
found that all mismatches either led to synonymous
substitutions or were present outside of annotated
features. Therefore, this plasmid was used to transform
into *M. extorquens*.

Following electroporation into *Mex*, colonies grew
on plates with the selectable marker tetracycline,
indicating successful insertion. Additionally, there
were positive results for the colony PCR using primers
for the assembled plasmid.

## Transformation of *tar* into *Mex*

---

Gibson assembly of the *tar* gene into pMW220 was
successful; the assembled plasmid is named pTAR001. We
succesfully transformed the *tar*-pMW220 plasmid
into *E.coli* DH5α, as evidenced by the positive
colony PCR (Figure 5), restriction digest (Figure 3), and sequencing
results (Figure 4). For the restriction digest, enzymes were chosen
which cut in both the insert and backbone to yield
meaningful band sizes.

![restriction digest of tar + pMW220 plasmids](https://static.igem.wiki/teams/5277/images/results3.webp)

**Figure 3 - a.) Restriction digest
of *tar* + pMW220 plasmids. b.) Expected
results for the restriction digests for assembled
plasmid (left) and empty backbone (right).** All
samples contained the restriction enzymes NheI-HF,
EcoRV-HF. The expected lengths of the bands if
assembly was successful were ~1kb,2.3kb and
9kb.

As seen from Figure 3, the plasmid in the first lane
(pTAR001) has the same results as that of the expected
bands for the assembled plasmid which suggests the
assembled plasmid has been correctly inserted. This is
confirmed by the sequencing results. The plasmids in
other lanes show improper band patterns, so we used the
plasmid in the first lane for all future
experiments. Sequencing confirmed a 100% identity of
this plasmid with our intended assembly.

![Sequencing results of pTAR001 compared to the expected result](https://static.igem.wiki/teams/5277/images/results4.webp)

**Figure 4 - Sequencing results of pTAR001
(bottom) compared to the expected result (top).**
pTAR001 is the plasmid used in the first lane of the
restriction digest.

Following electroporation into *Mex*, colonies grew
on plates with the selectable marker
gentamycin. Additionally, there were positive results
for the colony PCR.

![Colony PCR of pTAR001 in Mex.](https://static.igem.wiki/teams/5277/images/results5.webp)

**Figure 5 - Colony PCR of pTAR001
in *Mex.*** The expected band length was
805bp.

The colony PCR suggests that colonies 3, 4 and 6 contain
the pTAR001 plasmid. For future experiments, we chose to
use colony 3.

## Transformation of *lanM* + *tar* into *Mex*

---

We then transformed both the pLNM001 and pTAR001
plasmids into *Mex*. We did this via
electroporation. These transformations were reliably
successful, and were routinely confirmed by colony PCR (Figure 6).

![Colony PCR of pLNM001 and pTAR001 in mex](https://static.igem.wiki/teams/5277/images/results6.webp)

**Figure 6 - Colony PCR of pLNM001 and
pTAR001 in *Mex.*** The expected band length
was 845bp as pTAR001 was added to a colony that had
already been confirmed to have contained pLNM001 so
only primers for pTAR001 were added.

From figure 6, we can see that several colonies tested
had reliably uptaken the plasmid. We chose to use colony
3 as it had grown well and appeared successful on the
gel.

## Transformation of XoxF into BL21

---

![Colony PCR of pLNM001 and pTAR001 in mex](https://static.igem.wiki/teams/5277/images/results7.webp)

**Figure 7 - Transformed BL21 cells
containing pXOX001**

As seen in Figure 7, the pXOX001 successfully
transformed into BL21 cells.

![Colony PCR of transformed BL21 cells.](https://static.igem.wiki/teams/5277/images/results8.webp)

**Figure 8 - Colony PCR of transformed BL21
cells. 1-15 represent colonies from transformed plates,
16 represents the negative control, 17 represents the
positive control.** The expected band length was
921bp.

As seen in Figure 8, all the colonies from
transformation contained the gene insert xoxF, since the
bands aligned with the positive control. Though the
length is not that of the expected length, it was later
confirmed that this was an issue with the DNA
ladder. Therefore we grew the colonies and purified the
plasmids by miniprep to send off for sequencing at Full
Circle Labs.

![Sequencing](https://static.igem.wiki/teams/5277/images/results9-1.webp)
![Sequencing](https://static.igem.wiki/teams/5277/images/results9-2.webp)

**Figure 9 - Alignment of pXOX001
sequencing results with intended assembly**

## Transformation of *lutH* into *M. extorquens*

---

We attempted to transform our pUMP001 plasmid into
E. coli with heat-shock:

![transformde e coli that uptook the pump001 plasmid through heat-shock](https://static.igem.wiki/teams/5277/images/resultsu1.webp)

**Transformed E. coli that uptook the
pUMP001 plasmid through heat-shock.**

To test if our transformed bacteria had the *lutH*
insert, we conducted colony PCR.

![colony pct of transformed e.coli with luth insert](https://static.igem.wiki/teams/5277/images/resultsu2.webp)

**Colony PCR of transformed E. coli with *lutH*
insert.** 1-15 represent 15 colonies from our
transformation plate, 16 is the negative control
only containing pTE1877 without the insert and 17 is
the positive control of the Gibson assembly. The
expected band length was 674bp.

From the above figure, it can be seen that colonies 1-7
and colony 14 appeared to contain the insert, as the
bands somewhat aligned with the positive control. The
figure does however show that the amplicons are
heterogeneous, which indicated that there may
potentially be an issue. We grew the seemingly
successful colonies and purified them with miniprep to
send the plasmids containing *lutH* off for
sequencing at Full Circle Labs.

![](https://static.igem.wiki/teams/5277/images/resultsu3-1.webp)
![](https://static.igem.wiki/teams/5277/images/resultsu3-2.webp)

**Sequencing results for pUMP001 and pUMP002, which
are plasmids we purified using miniprep from
successful colonies containing pTE1877-*lutH*,
shown by colony PCR.**

The sequencing results in the above figure show that
only a part of the *lutH* gene was inserted into
the plasmid, suggesting that the bacteria have ejected
parts of the *lutH* gene because the pumps
expressed by this gene causes too much stress for the
bacterium, even under leaky, uninduced expression.

We would have chosen a different vector with a weaker
promoter, but around this time it became apparent
through modelling and WT characterisation that the WT
uptake was sufficiently efficient that
engineered *lutH* was unnecessary.

## Characterisation

## Wild type, *tar* and *lanM*+*tar* Swim plate assay demonstrating chemotaxis

---

We conducted swim plate assays to test our
chemotactically active engineered strains, *tar*
and *lanM* + *tar*, for chemotaxis towards
aspartate.

|  | Plate 1 | Plate 2 | Plate 3 |
| --- | --- | --- | --- |
| Wild type | ![](https://static.igem.wiki/teams/5277/images/char1-1.webp) | ![](https://static.igem.wiki/teams/5277/images/char1-2.webp) | ![](https://static.igem.wiki/teams/5277/images/char1-3.webp) |
| *tar* | ![](https://static.igem.wiki/teams/5277/images/char2-1.webp) | ![](https://static.igem.wiki/teams/5277/images/char2-2.webp) | ![](https://static.igem.wiki/teams/5277/images/char2-3.webp) |
| *lanM* + *tar* | ![](https://static.igem.wiki/teams/5277/images/char3-1.webp) | ![](https://static.igem.wiki/teams/5277/images/char3-2.webp) | ![](https://static.igem.wiki/teams/5277/images/char3-3.webp) |

**Table 1 - Swim plate assays of wild
type; *tar*; and *lanM* + *tar*
strain.** We did three repeats of each strain
represented at plate 1, 2 and 3.

The swim plates in Table 1 are composed of media and 0.2% w/v
agar. This was the required concentration to facilitate
motility in our strain, and unfortunately leads to the
agar splitting upon inoculation, reducing the robustness
of our data. However, the results can still be analysed
as below.

The more opaque circle in each picture is the 2% w/v
aspartate plug.

![Diagrammatic representation calculating the ratios of the distances between the inoculation point and the edge of the haze of bacteria. D1 represents the distance between the inoculation point and the point on the haze closest to the plug, and D2 represents the distance between the inoculation point and the point on the haze furthest from the plug. ](https://static.igem.wiki/teams/5277/images/char5.webp)

**Figure 1 - Diagrammatic representation calculating
the ratios of the distances between the inoculation
point and the edge of the haze of bacteria.** D1
represents the distance between the inoculation point
and the point on the haze closest to the plug, and D2
represents the distance between the inoculation point
and the point on the haze furthest from the plug.

The two distances in Figure 1, D1 and D2 (Figure 1), were measured
digitally on GeoGebra and their ratios were calculated
with the
formula: HazeRatio=D1D2
and the following results were obtained:

|  | Wild type | *tar* | *lanM* + *tar* |
| --- | --- | --- | --- |
| Plate 1 | 1.41 | 1.8 | 0.802 |
| Plate 2 | 1.054 | 1.898 | 3.563 |
| Plate 3 | 1.99 | 1.499 | 1.061 |
| Mean | 1.485 | 1.732 | 1.808 |

**Table 2 - Results of ratio calculations**

The split agar significantly impacted the reliability of
our data, such that we were unable to obtain statistical
significance with the number of replicates we
performed. We nonetheless consider the results to be
qualitatively promising, and would in future optimise
the setup to remove the noise generated by the
splitting.

| *tar* Plate 1 | *tar* Plate 3 | *lan* + *tar* Plate 2 |
| --- | --- | --- |
| ![tar plate 1](https://static.igem.wiki/teams/5277/images/char6-1.webp) | ![tar plate 3](https://static.igem.wiki/teams/5277/images/char6-2.webp) | ![tar plate 2](https://static.igem.wiki/teams/5277/images/char6-3.webp) |

**Table 3 - Swim plate assays showing chemotactic
response indicated with white arrows.**

*tar* Plate 1, *tar* Plate 3 and *lanM*
+ *tar* Plate 2 clearly showed that the cells swam
towards the aspartate plug indicating a chemotactic
response.

Therefore, we conclude that our strains with the
inserted gene *tar*, are likely to show more
chemotactic behaviour towards aspartate than the wild
type.

## Stress plate assay

---

This assay involves growing serial dilutions of cells on
media supplemented with increasing concentrations of
Nd. The range selected was intended to represent
concentrations around typical environmental conditions
linearly, and concentrations above environmental
conditions increasing exponentially. This was expected
to give the most informative dataset.

![Viability plates for WT mex after 3 days grown on variable concentrations of Nd](https://static.igem.wiki/teams/5277/images/spa1.webp)

**Figure 2 - Viability plates for
WT *M. extorquens* after 3 days grown on variable
concentrations of Nd**

Growth is supported at low concentrations of Nd, and is
preferred by the WT at 5ppm, so future assays were
conducted at 5ppm. Growth is inhibited at concentrations
as high as 10ppm, and almost prevented by 25ppm. No
growth is observed at higher concentrations (the visible
blemishes on the 1500ppm plate are caused by
NdCl3 diffusion into the 2µL droplet followed
by evaporation and precipitation of the salt).

Later in the project, when we had successful
transformants of lanM under inducible expression, we
repeated this experiment to demonstrate the difference
between the strains. The WT controls for this run of the
experiment were unfortunately contaminated, but we
present what results we do have for the *lanM*
strain. The lack of a control for these specific plates
sheds some doubt on these results, and we would like to
repeat this experiment with newly grown colonies.

![Viability plates for lanM strain](https://static.igem.wiki/teams/5277/images/spa2.webp)

**Figure 3 - Viability plates
for *lanM* strain.** IPTG is present to induce
expression.

These results can be quantified by counting colonies at
the lowest dilution for which growth can be observed,
and back-calculating the number cells that can viably
grow in the original OD600 = 1 culture.

![Viable cells in 100 against Nd concentration](https://static.igem.wiki/teams/5277/images/spa3.webp)

These results do not show the full range of stress for
LanM, but nonetheless demonstrate a clear success of
stress reduction over at least two magnitudes of [Nd].

## Neodymium uptake assay

---

This assay involved incubating cells in late
exponential phase overnight with IPTG and Nd. The
cells were then collected by centrifugation, lysed
by sonication and the resultant sample quantified
for Nd by ICP-MS. ICP-MS also facilitates
standardising to cell density by quantifying sulfur
present – an approximately constant average value
per cell in all our strains.

We incubated *M. extorquens* strains in 5ml
media containing 5ppm Nd. We tested WT and
inducible expression LanM transformants.

The volume of sample added to 5ml ICP-MS
(Inductively coupled plasma mass spectrometry)
solution (ultrapure 3.6% HNO3) was
calculated such that at 5ppm Nd (as for
equilibration by passive transport) the
concentration of Nd would be within the sensitive
range of ICP-MS: 5ppb-1000ppb.

| Sample | [Nd] in media (ppb) | [Nd] in pelleted sample (ppb) in 5ml HNO3 | [Nd] standardised by WT cell density (ppb) | [Nd] standardised to WT |
| --- | --- | --- | --- | --- |
| WT | 5000 | 9834 | 9834 | 1 |
| LanM | 5000 | 7162 | 14449 | 1.469 |

Consequently, this result is somewhat unreliable as
it lies outside of the calibration curve, but the
magnitude remains reasonably reliable.

Clearly, the uptake at this concentration is already
extremely efficient even in the WT. Consequently, we
decided against continuing to engineer in uptake
mechanisms, and focused on stress mitigation for the
bioaccumulation aspect of the project.

We do see a noticeable increase in uptake in
the *lanM* strain, which indicates that our
idea of using it as a buffer system to sequester
neodymium from neodymium-inhibited uptake pathways
is functional.

## Wild type *Mex* chemotaxis swim plate characterisation (unsuccessful)

---

We conducted swim plate assays with wild type
*Methylobacterium extorquens* to determine if the
wild type strain already has a chemotactic response to
chemoattractants for which *E. coli* shows positive
taxis such as aspartate, methanol and other chemicals
such as maltose, serine and galactose.

| Buffer only plug (negative control) | Aspartate plug (or other test attractant) | Methanol plug (positive control) |
| --- | --- | --- |
| ![1](https://static.igem.wiki/teams/5277/images/charun1-1.webp) | ![2](https://static.igem.wiki/teams/5277/images/charun1-2.webp) | ![3](https://static.igem.wiki/teams/5277/images/charun1-3.webp) |

**Swim plate assays of Wild Type
M. extorquens with media only plug (negative control),
aspartate plug and methanol plug.**

As seen in the above table, *M.extorquens* appears
not to show chemotactic behaviour towards any of the
plugs, including the positive control, indicating an
unsuitable experimental setup. We optimised in several
ways, including by decreasing the concentration of agar
in the plates to better facilitate motility, but we were
able to obtain no positive results, even for the
positive control methanol.

From this experiment we could not conclude whether the
wild type strain of M. extorquens had a chemotactic
behaviour or not towards the plugs. Therefore, we
redesigned the setup to use a growth supportive media,
which yielded results that could be more easily
troubleshot.

| Buffer only plug (negative control) | Aspartate plug (or other test attractant) | Methanol plug (positive control) |
| --- | --- | --- |
| ![1](https://static.igem.wiki/teams/5277/images/charun2-1.webp) | ![2](https://static.igem.wiki/teams/5277/images/charun2-2.webp) | ![3](https://static.igem.wiki/teams/5277/images/charun2-3.webp) |

We saw growth in these plates, but no motility, which
was very concerning, because M. extorquens is widely
characterised as being motile
([Tsagkari E, Sloan W, 2018](https://www.mdpi.com/2073-4441/10/10/1386))
([Alessa O et al., 2021](https://www.ncbi.nlm.nih.gov/pubmed/34737731)). We
concluded that the strain we had received from the
Tobias Erb lab was likely domesticated such that it had
lost motility, which is a documented phenomenon
([Barreto H et al., 2020](https://www.ncbi.nlm.nih.gov/pubmed/33144634))
([Patrick J, Kearns D, 2009](https://www.ncbi.nlm.nih.gov/pubmed/19749039)). As
a result, we began to perform a directed evolution
experiment, wherein we took cells from the farthest
point they had reached from inoculation, and regrew them
on new swim plates.

Repeating this several times (2-3) produced more motile
*M. extorquens*, which displayed an unusual
characteristic motility – it would migrate to the edge
of the plate, then spread radially from there. This
could perhaps be due to a clumping or quorum sensing
pathway. This phenotype is not dissimilar to the
phenotype for *Methylobacterium* demonstrated
[here](https://www.mdpi.com/2073-4441/10/10/1386).

In any case, we reran the growth-supportive swim plates
with attractant-in-plug, but saw no motility – we have
come to the conclusion that the primary metabolic
substrate methanol induces motility, as the cells have
more energy available. Methanol could be used in the
directed evolution swim plates, but was excluded from
the attractant-in-plug taxis plates because it would
have interfered with the positive control.

As a result, we ordered the DNA for the
aspartate-sensing MCP Tar, and decided to rerun the swim
plates with methanol using aspartate as the attractant,
as has been detailed in the successful results section.

## Proof of Concept

## Lanthanide enrichment proof of concept swim plates

---

Our proof of concept aimed to see if our bacteria could
enrich certain spatial areas in Nd. That is, we want to know
if our bacteria disrupt homogeneity of Nd within the
media. To this end, we collected samples from 3 distances
from the plug, and quantified the Nd in them. Before this,
however, we were able to define our statistical null
hypotheses.

H0, homogeneity: The concentration of Neodymium
in the media is **uniformly distributed**.

H0, enrichment: The concentration of Neodymium in
the media is **uniformly distributed**, with **µ = [Nd]
added to plates**.

The purpose of H0, homogeneity is to capture
whether we see a disruption in the uniformity of Nd within the
locations of our samples.

The purpose of H0, enrichment is to capture
whether we see a different concentration of Nd than expected
by our calculated value for [Nd] within the plates. This is
useful if the bacterial and thus neodymium migration
patterns are not accurately represented by our sample
collection methodology.

We represent the results visually in a histogram-reminiscent
graph (Figure 1). (Full data is available supplementarily.) These
graphs display the average Nd across the range of distance
from the centre of the plug, which we believe to be the most
scientifically precise visualisation with our sample
collection methodology.

![figure 7-1](https://static.igem.wiki/teams/5277/images/char7-1.webp)
![figure 7-2](https://static.igem.wiki/teams/5277/images/char7-2.webp)
![figure 7-3](https://static.igem.wiki/teams/5277/images/char7-3.webp)
![figure 7-4](https://static.igem.wiki/teams/5277/images/char7-4.webp)

**Figure 1 - Spatial visualisation of Nd.** Top left:
WT. Top right: *tar*. Bottom left: *lanM tar*
uninduced, Bottom right: *lanM tar* induced.

Performing Chi-squared statistical tests on these datasets
yields the following results:

µ for H0, enrichment is for these plates
calculated as = 4.167 ppm. A concentration of 5ppm was added
to the plates, which was subsequently diluted by addition of
1/5 volume of a solubilisation buffer after sample
collection.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | H0, homogeneity | H0, enrichment | Notes || WT | p = 0.707 | p = 0.590 |
| tar | p > 0.999 | p = 0.533 |
| *lanM tar* uninduced | p = 0.987 | p = 0.304 |
| *lanM tar* induced | p = 0.181 | p = 0.003 < 0.05 | One replicate appeared to fail – excluding this yields p = 0.0280 < 0.05 for H0, homogeneity. It is poor practice to exclude replicates without cause, so we mention this but do not claim this level of significance. |

We see what we consider to be a likely; and biologically and
industrially meaningful disruption of homogeneity, but we do
not achieve strict statistical significance by this metric
with only duplicates – a limitation of ICP-MS is that the
cost prevented us running more samples.

However, it is probable that we miss Nd migration from the
very edges of the plate with our collection methodology,
because we do see a statistically significant greater set of
values than expected across the board in our complete,
induced strain. The greater than expected neodymium
concentrations in these samples must have come from
somewhere, and the most reasonable explanation is that the
neodymium came from the edges of the plate.

In our proof of concept, we do also see a clear trend in
[Nd], increasing closer to the plug, as expected.

We do not see effective Nd migration in the *tar* only
strain, and only very minor, statistically insignificant
migration in the uninduced *tar*, *lanM*
strain. We hypothesise that this is a consequence of that
cells that uptake Nd immediately begin to express Tar, and
therefore swim towards the plug, which drastically reduces
the amount of Nd they take with them.

We believe LanM acts as a buffer against uptaken Nd,
preventing it from mediating an upregulation in *tar*
expression until the LanM is saturated and cannot sequester
any additional Nd.

[Supplementary data for proof of concept](%7B%7B%20url_for%28%27static%27%2C%20filename%3D%27pocsupdat.xlsx%27%29%20%20%20%7D%7D)

## ICP-MS

## What is ICP-MS?

---

**Inductively Coupled Plasma Mass Spectrometry
(ICP-MS)** is an advanced analytical technique used
to detect and quantify trace elements and isotopes in
a variety of samples. It works by converting a liquid
sample into an aerosol, which is then introduced into
a high-temperature plasma (approximately 6,000 to
10,000 Kelvin) generated by argon gas. In the plasma,
the sample undergoes ionization, transforming the
atoms into positively charged ions. These ions are
then directed into a mass spectrometer, where they are
separated based on their mass-to-charge ratio (m/z). A
detector measures the intensity of each ion, providing
a highly sensitive analysis of the sample's elemental
composition with detection limits often in the
parts-per-billion (ppb) or parts-per-trillion (ppt)
range.

---

### Why are we using it?

Aside from it being a very cool technique (that the
chemist in our team really geeked out on), the
significance of ICP-MS in our project lies in its
ability to accurately measure the uptake of neodymium
(Nd) by our genetically engineered cells compared to the
wild-type strains. Nd is a rare earth metal that is not
abundantly present in biological systems, and detecting
its precise concentration is crucial to assessing the
efficacy of our bioremediation or bioextraction
processes. By using ICP-MS, we can quantify even minute
differences in Nd accumulation within our modified
cells, providing a clear picture of how genetic
modifications impact the ability of our bacteria to bind
or absorb neodymium. This comparison is essential for
validating whether our engineered strains exhibit
enhanced metal uptake over their unmodified
counterparts, which is a key indicator of the success of
our project.

The analytical process begins by lysing the bacterial
cells to release any accumulated Nd into the liquid such
that it can become homogenous. Once prepared, the sample
is introduced into the ICP-MS, where the Nd ions are
ionized in the plasma and directed into the mass
spectrometer for separation and detection. Because
ICP-MS is highly sensitive and can distinguish between
different isotopes, it allows us to specifically
quantify Nd in the presence of other elements, even if
they are present in much higher concentrations. This
specificity is vital for our research, as it enables us
to accurately monitor Nd uptake without interference
from other components of the bacterial growth medium or
residual elements present in the cells.

Moreover, ICP-MS can provide isotope ratio information,
which could be used to trace the origin of Nd if
desired. In our context, while this feature may not be
directly relevant for uptake studies, it could become
important if we want to differentiate between Nd sourced
from different electronic waste materials, or if we wish
to track different Nd supplementation in the lab. The
technique's speed and ability to handle complex matrices
also make it particularly suitable for processing
multiple samples efficiently, which is necessary for
comparative studies between wild-type and engineered
strains.

Finally, one of the most significant aspects of using
ICP-MS for our project is its role in refining and
validating our biotechnological approach. By generating
robust quantitative data, ICP-MS helps us establish the
relationship between genetic modifications and
metal-binding efficiency. This enables us to iteratively
improve our strains, making ICP-MS not just a tool for
measurement but a cornerstone of our experimental
validation and strain development process. Through
repeated testing and analysis, we can confidently
determine whether our engineered cells outperform the
wild type, ultimately supporting our goal of developing
a sustainable and efficient method for rare earth metal
recycling.

---

### Protocol

We collected various types of samples, containing cells
throughout our project. Cells were always lysed by
sonication to allow neodymium to become homogenous in
the samples.

After lysis, the cell samples were allowed to cool to
room temperature. In a fume hood, 5 mL of 3.6% nitric
acid (HNO₃) was added to each lysed sample to digest the
cellular material and release any bound neodymium. The
mixtures were vortexed thoroughly and left to incubate
at room temperature for 1 hour to ensure complete
digestion of the cellular matrix. Following the
digestion period, deionized water was added to each
sample, bringing the final volume to 5 mL, which
resulted in a final nitric acid concentration of
approximately 10%. The prepared samples were then
transferred to clean ICP-MS vials for subsequent
analysis.

Before introducing the samples to the ICP-MS instrument,
the system was calibrated using standard solutions of
neodymium at known concentrations to establish a
baseline for accurate quantification. In addition to the
experimental samples, blank samples (3.6% nitric acid in
deionized water) and quality control standards were
included to ensure reliability and precision throughout
the analytical run. The samples were then introduced
into the ICP-MS using the automated sample introduction
system, where the instrument separated the ions based on
their mass-to-charge ratio and quantified the Nd content
using isotopic signatures such as 144Nd
or 146Nd. The results provided a precise
measurement of neodymium concentration within each
sample, allowing for direct comparison between the
engineered strains and the wild-type bacteria.

Post-analysis, the ICP-MS system was thoroughly flushed
with deionized water to remove any residual nitric acid
or metal ions, preventing contamination in subsequent
runs. All waste generated, including the acid-digested
samples and rinsing solutions, was disposed of following
appropriate hazardous waste protocols to ensure
environmental safety and compliance with laboratory
regulations. By following this protocol, the total Nd
content per gram of dry cell mass or per gram of sample,
as appropriate, was accurately determined, providing a
clear assessment of the efficiency of Nd uptake by the
genetically modified strains compared to the
wild-type. This data is crucial for evaluating the
success of our project’s aim to enhance rare earth
element recovery through bioengineering.

---

### Explaining our results

In our initial set of ICP-MS measurements, we assessed
the neodymium (Nd) uptake capacities of our wild-type
(WT) *Methylobacterium extorquens* (*Mex*)
strain and the genetically modified strain expressing
the LanM protein (*lanM* strain). The ICP-MS
analysis revealed a significantly higher concentration
of Nd in the *lanM* strain compared to the
wild-type. This result indicates that the LanM protein,
which is designed to selectively bind and sequester
lanthanides, substantially enhances the bacteria’s
ability to accumulate neodymium from the surrounding
medium.

The increased Nd concentration in the *lanM* strain
confirms the functional role of the LanM protein in
facilitating the uptake of rare earth elements (REEs)
such as neodymium. The wild-type strain, lacking this
specific protein, showed relatively lower Nd
concentrations. This disparity between the two strains
is crucial, as it validates our genetic engineering
approach to improving REE bioaccumulation and suggests
that the LanM protein could be a valuable tool for
bioremediation applications.

To confirm the difference in Nd concentrations between
strains, we performed calculations to standardize our
data. Due to low sensitivity, we were unable to use
Sulfur and phosphorus to generate reliable calibration
curves (Figure 1). We thus also were not able to use
these two elements in our data and get absolute values
for Nd concentration **per individual cell**. In
turn, we had to use copper (Cu) due to sensitivity
towards it being significantly higher. This should be
accurate but prevents the calculation of [Nd] per
individual cell, so only
**relative values per cell density** can be obtained.

![copper [He] calibration graph](https://static.igem.wiki/teams/5277/images/micpms11.webp)
![Neodymium [no gas] calibration graph](https://static.igem.wiki/teams/5277/images/micpms12.webp)

**Figure 1 - ICP-MS calibration curves for A) 63 Cu
[He] and B) 144 Nd [No gas].**

Despite not having absolute cell mass, we could still
standardize relatively to cell density accurately. The
calculations we performed are as follows:

---

### Calculations

Where x = the concentration of 144 Nd [No gas] from
graph (ppb):

144 Nd [No gas] Signal Intensity (CPS)=39038x−92937

x=144 Nd [No gas] Signal Intensity (CPS)+9293739038

The same was done for the calibration curve using Cu
(see Figure 1). The following results were obtained:

| Samples | 144 Nd [No gas] CPS | Conc. of 144 Nd [No gas] from graph (ppb) | ppb from instrument |
| --- | --- | --- | --- |
| WT – 1 | 569672284,8 | 10699,90998 | 9169,448 |
| WT – 2 | 671578990 | 12613,47124 | 10499,281 |
| LanM – 1 | 546952725,2 | 10273,29164 | 8539,971 |
| LanM – 2 | 342690728,2 | 6437,745737 | 5783,934 |

| Samples | 63 Cu [He] CPS | Conc of Cu 63 [He] from graph (ppb) | ppb from instrument |
| --- | --- | --- | --- |
| WT – 1 | 177,33 | 7,340408834 | 5,374 |
| WT – 2 | 240 | 9,682424605 | 7,598 |
| LanM – 1 | 109,67 | 4,811913749 | 2,794 |
| LanM – 2 | 138 | 5,870622968 | 3,636 |

We then calculated averages of copper to get the
relative ratio of cell density. This was then used to
standardise concentrations of Nd by cell density. It is
assumed that Cu is directly proportional to cell number
and constant between strains.

| Samples | Average Cu (ppb) | Standardised to WT |
| --- | --- | --- |
| WT | 6,486 | 1 |
| LanM | 3,215 | 0,49568301 |

| Samples | [Nd] in pelleted sample (ppb) in 5mL HNO3 | [Nd] standardised by WT cell denisity (ppb) | [Nd] standardised to WT |
| --- | --- | --- | --- |
| WT | 9834 | 9834 | 1 |
| LanM | 7162 | 14449 | 1,469 |

## Enzymatic Assay for Neodymium

## XoxF Protein Purification

---

We put a lot of work into attempting to purify XoxF, but
we were unfortunately unsuccessful due to the lack of
experience with protein work across the team.

Initially, we were directed to the expression vector
pET-28a(+) for *E. coli* BL21. We successfully
assembled and transformed this plasmid through DH5α into
BL21. However, upon purification it became apparent that
the protein was retained in the insoluble fraction, as can
be seen in the SDS gel below.

![](https://static.igem.wiki/teams/5277/images/eafn1.webp)

**SDS-PAGE of XoxF purified from *E. coli* BL21
using the expression vector pET-28a(+).** Ladder
used is Thermofisher Pageruler Prestained Ladder. The
expected band value for purified protein is 66kDa.

This gel strongly indicates that the protein is insoluble
in our expression system. Unsurprisingly, this prevented
it from showing any activity. Had we had more experience
with protein work on the team, we would have switched our
expression system to a successful purification from the
literature at this stage. However, we did not recognise
the issue posed by the protein being insoluble.

Therefore, we attempted several alternative conditions to
promote solubilisation, including:

* Using lysate directly.
* Reduced concentration of inducer (IPTG).
* Performing the assay *in vivo*.

Unfortunately, none showed activity – it is likely the
protein was aggregated in inclusion bodies in every
attempt.

At this stage, the iGEM deadline was fast approaching, so
we did not have time to redesign our expression system. We
therefore attempted a solubilisation and refolding
protocol for inclusion bodies. Unfortunately, we lacked
some of the requisite chemicals and materials for
efficient refolding, and were therefore unable to produce
active purified protein. We did reliably obtain denatured,
solubilised protein at a reasonable concentration, as
shown by the Nanodrop below (Figure 1).

![nanodrop a280 protein in urea based denaturing solution](https://static.igem.wiki/teams/5277/images/eafn2.webp)

**Figure 1 - Nanodrop A280 of protein in Urea
based denaturing solution.**

## Neodymium Quantification Assay Using XoxF

---

Unsurprisingly, without active protein our enzymatic assay
could not be performed. We present a calibration curve for
our photometrically active chemical DCPIP under the
conditions of use (Figure 2), and deeply regret that we cannot
present results for the assay itself.

![calibration curve for DCPIP at pH 8.8 at room temperature](https://static.igem.wiki/teams/5277/images/eafn3.webp)

**Figure 2 - Calibration curve for DCPIP at
pH 8.8 at room temperature.**  Data points are
triplicated. Error bars represent standard deviation.

DCPIP does not mix rapidly with water, and therefore
requires thorough mixing by pipetting before readings are
taken.

The gradient 0.0131 is the molar extinction coefficient,
ε, in µM cm-1. This can be converted to ε =
13.1 mM cm-1, which is comparable to that in the
literature, as
summarised [here](https://www.ncbi.nlm.nih.gov/pubmed/32060650).

{% endblock %}