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Lab Journal

Laboratory Timeline

June 16th-25th: Generation of a recombinant E. coli strain for heterologous production of LanM

1) Cultivation of the natural LanM producer Meythlorubrum (M.) extorquens AM1 on agar plates (hypho medium). This served as a potential source for genomic DNA, templating for amplification of the lanM gene.

2) Molecular cloning of pET-19b(+)::lanM. The M. extorquens AM1 strains grew very slow, so already present M. extorquens PA1 genomic DNA was used to amplify lanM. Following the NEBuilder® workflow, the insert was cloned into the high expression vector pET-19b(+). The ligated product was validated by colony PCR of transformant E. coli DH5α strains and by Sanger sequencing.

3) Generation of the final LanM expression host. To generate the final LanM expression host, pET-19b(+)::lanM was transformed into E. coli BL21(DE3). This strain was cultivated in LB in the first LanM production run.

June 29th - July 13th: Verification of LanM production, development of simplified LanM purification protocol, and large scale LanM production

1) An SDS-PAGE was run with the crude cell extract of E. coli BL21(DE3) pET-19b(+)::lanM to investigate if the strain produced recombinant LanM. Indicated by a strong band at the expected protein size (13 kDa), the production system showed successful biosynthesis of the desired protein.

2) To circumvent costly and time-consuming affinity chromatography for LanM purification, two alternative approaches were tested. One process applied acidification of the crude cell extract to remove all proteins except LanM. The other approach made use of the putatively higher heat resistance of LanM compared to E. coli proteins, enabling the denaturation of non-LanM proteins to obtain pure LanM. The protein pattern of the supernatant (non-precipitated proteins) was checked for both approaches via SDS-PAGE. The heat-based process showed promising results, albeit some proteins were present besides LanM after heating up to 95 °C. In contrast, a pH reduction to pH 2.5 removed all proteins except LanM.

3) LanM was produced in a large scale (1 l E. coli culture) to provide enough of this biomolecule for upcoming experiments.

August 4th-25th: Options to precipitate LanM from solution

1) Ammonium sulfate: It was assessed at which ammonium sulfate concentration LanM precipitates. Initial results indicated that an elevated concentration is necessary.

2) Ethanol: The second option for LanM precipitation relied on the addition of the protein solution to cold ethanol (-20 °C) and an incubation at -80 °C (1 h). This process was successful as well, although only initial experiments were conducted and final evaluation of precipitation yields was still due.

In addition, it was still to be clarified if the chelating capacity of LanM is impaired during exposure to ethanol.

August 26th - September 1st: Options to precipitate LanM from solution

The XO assay comprises the dye xylenol orange which undergoes an absorption shift to 574 nm when it binds to certain metal cations, including rare earth elements (REE). When LanM is present in the same assay, it serves as the preferred chelating agent until its REE binding sites are saturated.

This assay was established and tailored to the required analytical application. Calibration was performed in the absence of LanM. After adding LanM, the REE buffering capacity of this protein was determined.

September 1st-29th: Running the first process for LanM-based REE extraction from ores

1) Following LanM purification and apatite ore leaching, the whole process was run in its first design. It comprised the isolation of the LanM-REE complex via salting-out with ammonium sulfate. During the process, precipitates formed at various stages. After conducting a control run without LanM and precipitate formation after ammonium sulfate addition continued, it was concluded that salting-out led to an unspecific coprecipitation of mineral salts. Thus, the process design was to be refined.

2) XO assay: It was assessed if XO shows a color change in the presence of non-REE cations abundant in the apatite ore (Ca2+, Na+ and Fe2+). Only for ferrous ions, an increase in A574 was detected, albeit demanding the presence of elevated concentrations.

September 30th - October 12th: Running the improved process for LanM-based REE extraction from ores, including several alterations.

1) The salting-out step was replaced by an ultrafiltration-based separation of the LanM-REE complex from the ore leachate. After running this process once, all derived solutions (flow-throughs, retentates) were assessed with respect to the cation contents. First XO assay-based results supported the expectation that the redesigned process was capable of REE isolation. ICP-MS was performed to solidly investigate the cation abundance pattern.

2) The redesigned process was additionally run in the absence of LanM (no-protein control) to investigate the dependence of REE extraction on LanM. Deduced from XO assays, almost no REEs were present in the final ultrafiltration flow-through fraction if LanM was absent, in contrast to the high abundance of these cations if LanM was applied in the process.

3) To enable rapid determination of protein contents, e.g. for tracing of LanM migration through ultrafiltration membranes or for determination of LanM amount-specific REE extraction yields, the BCA protein determination assay was used. It was shown, e.g. that LanM does not pass the ultrafiltration membrane in a substantial extent.

4) An apatite leachate sample was subjected to ICP-MS-based analysis of cation composition.

5) A second REE extraction cycle was performed with LanM retained from a first extraction run to test the reusability of the biomolecule.

6) Instead of applying ore as feedstock, synthetic REE-phosphates (LaPO4, LuPO4) were employed in a final whole-process investigation. This aimed at comparing the process suitability for extraction of heavy REE vs. light REE.

7) Ammonium sulfate- and ethanol-based precipitation behavior of LanM was precisely assessed. Obtained LanM yields, i.e. percentage of applied LanM retained in the pellet, were determined. LanM activity after ethanol treatment was determined.