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<a href="#ConstructionofthemutantlibraryofOsNramp5">Construction of the mutant library of
OsNramp5</a>
<a href='#Screeningoferror-pronePCR'>Screening of error-prone PCR random mutation library</a>
<a href="#Cadmiumrespondingbiosensor">Cadmium responding biosensor</a>
<a href="#Bioinformatics-assistedmutationsitesselection">Bioinformatics-assisted mutation sites
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<h1 style="margin:4vh 0;">Overview</h1>
<p style="font-size: 1.5em;"><b> Construction of the mutant library of OsNramp5</b></p>
<p><b>Establishment of error-prone PCR experimental system</b></p>
<p><b>Construction of the random mutation library with homologous recombination</b></p>
<p style="font-size: 1.5em;"><b>Screening of error-prone PCR random mutation library</b></p>
<p><b>Determination of Cd<sup>2+</sup>concentration in solid media</b></p>
<p><b>Screening of OsNramp5-mut strains transferring less Cd<sup>2+</sup></b></p>
<p><b>Screening of <i>OsNramp5-mut</i>strains transporting Mn<sup>2+</sup> normally</b></p>
<p style="font-size: 1.5em;"><b>Cadmium responding biosensor</b></p>
<p style="font-size: 1.5em;"><b>Bioinformatics-assisted mutation sites selection</b></p>
<p><b>Prediction of OsNramp5 protein topological structure</b></p>
<p><b>Prediction of OsNramp5 protein mutation sites</b></p>
<p style="font-size: 1.5em;"> <b>Reference</b></p>
<a id='ConstructionofthemutantlibraryofOsNramp5' class="target-fix"></a>
<h2>Construction of the mutant library of OsNramp5 </h2>
<p><b>Establishment of error-prone PCR experimental system. </b></p>
<p>Recombinant plasmid pFL61-OsNramp5 was constructed by homologous recombination. The mutation
frequency of the library was positively correlated with the amount of StarMut Enhancer added into
the reaction system. The reaction system contained
0, 1, 2.5, 5, 10, 20 μL StarMut Enhancer to obtain PCR amplification of OsNramp5 cDNA sequence.
According to the result of electrophoresis, 10 μL StarMut Enhancer performed the best result. The
mutant ratio is about 6.5‰ when 10 μL
StarMut Enhancer is added into the reaction system, according to kit instructions. The optimum
temperature of error-prone PCR is 63.2 ℃ according to the gradient experiment. </p>
<div class="content_img">
<img src="./images/result1.jpg" alt="" class="result_viewer">
<p><b>Fig.1 Error-prone PCR results at gradient temperature. </b> Minimal dispersion can be observed
at 63.2 °C, while the brightness of the bands indicated a more successful amplification.
Therefore, the annealing temperature was set
as 63.2 ℃ in the subsequent experiments. </p>
</div>
<p><b>Construction of the random mutation library with homologous recombination. </b></p>
<p>Linearized pFL61 plasmids and OsNramp5 mutant sequences were used to conduct homologous recombination
and built a mutant plasmid library. After the plasmid library was constructed, yeast transformation
was performed and screening could
be carried out using SD-Ura solid media. Thirteen rounds of plasmids were transformed and about 200
plates were plated in 6 weeks. After 72 hours of incubation in 30 ℃, positive transformants were
re-delineated. Twenty-one library
plates (Fig.2) were established with a total capacity of about 260 colonies. During our research, we
found that the homologous recombinant plasmids showed instability in heredity and low efficiency in
the process of transformation.
The plasmids also came out with the problem during PCR. We could observe smears or loss of our
target bands. We supposed that the simple homologous recombination process lacks the common
methylation and phosphorylation which happen
normally in common cells, thus decreasing the stability of the plasmids and leading to their
breakage during PCR. </p>
<p>Afterwards, to solve this problem, we made attempt to transform the mutant recombinant plasmids into
<i>E.coli (Escherichia coli) </i> DH5α for amplifying firstly, and then transferred them into
<i>Saccharomyces cerevisiae Δycf1</i> .The results revealed that the efficiency of transformation
was greatly improved. The plates covering 100μL yeast solution showed up to almost 200 CFUs per
plate, far exceeding the previous 2 to
30 CFUs. However, after the plasmid amplification in
<i>E.coli</i> the number of the same yeast clones turned out to be huge as well. 115 strains were
selected randomly to fill in the mutation library.
</p>
<div class="content_img">
<div style="white-space: nowrap;">
<img src="./images/result2.jpg" alt="" class="result_viewer"
style="width: auto;height: 20vh;display: inline-block;">
<img src="./images/result3.jpg" alt="" class="result_viewer"
style="width: auto;height: 20vh;display: inline-block;">
</div>
<p><b>Fig. 2 Part of the mutant libiaries. </b></p>
</div>
<a id='Screeningoferror-pronePCR' class="target-fix"></a>
<h2>Screening of error-prone PCR random mutation library</h2>
<p><b>Determination of Cd<sup>2+</sup>concentration in solid media. </b></p>
<p> Solid SD-Ura media were set up with different Cd<sup>2+</sup> concentration: 0, 30, 40, 50, 100 μM,
to find a proper Cd<sup>2+</sup>concentration. We tried to get a concentration at which the
phenotype of yeast transferred to unedited
pFL61 plasmid as control group was significantly better than that in <i>HR</i>, the yeast containing
<i>OsNramp5-ori</i>. The solid media with too much Cd
<sup>2+</sup> led to poor phenotype for <i>pFL61</i> and <i>HR</i>, while the low Cd<sup>2+</sup>
concentration in the solid media results in good growth for control and they can't make any
difference. In the two occasions mentioned
above, we can't decide if the phenotype of yeast in experiment group (<i>OsNramp5-mut</i> yeast) is
close to that of <i>Saccharomyces cerevisiae</i> that we inserted in <i>pFL61</i> or <i>HR</i>
(<i>OsNramp5-ori Saccharomyces cerevisiae</i>).
Yeasts with a similar phenotype to <i>pFL61</i> are defined as potential benign mutants while yeasts
with phenotypes similar to or worse than <i>HR</i> are considered to contain deleterious mutation
sites or null mutation sites. The
phenotype difference between <i>pFL61</i> group and <i>HR</i> group is the most evident when the
concentration of Cd<sup>2+</sup> is 30 μM. The growth state is suitable for observation after being
cultured for 72h. Therefore, the concentration
of Cd<sup>2+</sup> in the solid SD-Ura media was set as 30 μM in the following experiment.
</p>
<div class="content_img">
<img src="./images/result4.jpg" alt="" class="result_viewer">
<p><b>Fig.3 The growth of yeast at different Cd<sup>2+</sup> concentrations. </b> The most suitable
concentration for screening potential benign mutant strains was 30 μM. </p>
</div>
<p><b>Screening of OsNramp5-mut strains transferring less Cd<sup>2+</sup>.</b></p>
<p>Recombinant plasmids were transformed into yeasts, and they could not grow on the solid SD-Ura media
without this kind of plasmid. In the process of library construction, we got 260 positive
transformants totally. Then, the single colony
of each transformant was propagated in liquid SD-Ura media. Different densities of yeast were
obtained by gradient dilution, to spot on SD-Ura solid media containing Cd<sup>2+</sup>. By doing
so, the effect of different yeast concentration
was further avoided on the final phenotypic observation. 6 mutant strains (Fig.4) were selected by
spot experiments, of which the phenotype was close to the control group. Therefore, they were
recognized as potential benign mutants.
These mutants were further sequenced and analyzed, in order to explore associations among these
mutation sites in different mutants. Besides, we also obtained mutants with poorer phenotypes than
<i>HR</i>.They may serve as hypersensitive strains of cadmium and may be useful in other future studies on cadmium stress. We concluded that OsNramp5
of these mutants probably increased their ability to transport Cd<sup>2+</sup>. Therefore, the
mutation sites of these mutants were probably related to Cd<sup>2+</sup> transportation. </p>
<p>Six mutants with benign phenotype we selected were sequenced, and sequences of two mutants were
successfully obtained. Some mutation sites were finally found in OsNramp5 of ep-17: H21L, P22A,
Q23P, K26Q, L78Q; in OsNramp5 of ep-11: Q78L,
S162G, Q167L, T294A. Due to frame shift mutation, ep5-11 was terminated early at 298<sup>th</sup>
site and ep5-17 was terminated early at the 114
<sup>th</sup> site. We analyzed that the structure of the protein was damaged resulting in the loss
of its ability to transport cadmium, and this change was likely to also affect the transport of Mn
<sup>2+</sup>.
</p>
<div class="content_img">
<div style="white-space: nowrap;">
<img src="./images/result5.jpg" alt="" class="result_viewer"
style="width: auto;height: 20vh;display: inline-block;">
<img src="./images/result6.jpg" alt="" class="result_viewer"
style="width: auto;height: 20vh;display: inline-block;">
</div>
<div style="white-space: nowrap;">
<img src="./images/result7.jpg" alt="" class="result_viewer"
style="width: auto;height: 20vh;display: inline-block;">
<img src="./images/result8.jpg" alt="" class="result_viewer"
style="width: auto;height: 20vh;display: inline-block;">
</div>
<p><b>Fig.4 Potential benign mutant strains ep 5-11 and epⅠ. </b> The growth of ep 5-11 and epⅠ on
SD-Ura plates contains Cd<sup>2+</sup> concentration of 30 μM. It was slightly better than that
of <i>pFL61</i> and <i>HR</i> while they
grew similarly to <i>pFL61</i> and <i>HR</i> on plates without Cd<sup>2+</sup>, indicating that
ep 5-11 and epⅠmay be the potential OsNramp5-mut strains. Its ability of transporting
Cd<sup>2+</sup> has successfully decreased or
their OsNramp5 has been damaged. </p>
</div>
<p><b>Screening of <i>OsNramp5-mut</i> strains transporting Mn<sup>2+</sup> normally. </b></p>
<p>The mutants with reduced transport of Cd<sup>2+</sup> selected in primary screening were treated as
new experimental materials. They were spotted on Mn<sup>2+</sup>-deficient SD-Ura media with the
control group (<i>Δsmf1-pFL61</i> and
<i>Δsmf1-HR</i>). The Mn<sup>2+</sup> in media was specifically chelated by EGTA. As for the mutant
strains, <i>Δsmf1-pFL61</i> could not grow well in the media while <i>Δsmf1-HR</i> can, because the
<i>Δsmf1</i> strain cannot transport
Mn
<sup>2+</sup> normally, they already had an additional Mn<sup>2+</sup> OsNramp5 transporter whose
ability of transporting Cd<sup>2+</sup> is decreased. If its ability to transport Mn<sup>2+</sup> is
not affected by the mutation, its
growth phenotype should be similar to <i>HR</i>. If the function of OsNramp5 in transporting
Mn<sup>2+</sup>is damaged during the mutation, its growth phenotype should be similar to that of
<i>pFL61</i>.
</p>
<p>The re-screening strategy using Mn<sup>2+</sup> was exactly the same as the primary screening by Cd
<sup>2+</sup>. Series of densities of yeast solution was set for spot experiment. In conclusion, the
strategy to select the benign OsNramp5-mut which can transport Mn<sup>2+</sup> normally is that the
closer the phenotype of the mutant
is to <i>HR</i>, the more likely it is to be the benign mutant strain we expect. Due to the time
limitation, it was difficult for us to obtain this part of the data before the paper was submitted,
but relevant experiments have been
conducted. If the experiments go smoothly, we hope to be allowed to present our final screening
results in the thesis defense.
</p>
<a id='Cadmiumrespondingbiosensor' class="target-fix"></a>
<h2>Cadmium responding biosensor </h2>
<div class="content_img">
<img src="./images/result9.jpg" alt="" class="result_viewer">
<p><b>Fig.5 Effect of Cd<sup>2+</sup> concentration on engineered bacteria. </b></p>
</div>
<p>Researchers have found that some microorganisms show good ability in Cd<sup>2+</sup> resistance and
removal (Albano & Macfie, 2016). Especially, <i>Pseudomonas aeruginosa</i> is a heavy-metal
resistant bacteria isolated from industry wastewater,
which possesses high resistance to Cd
<sup>2+</sup>. Further researches showed that the Cd<sup>2+</sup> resistance ability relies on two
metal-recognizing regulatory proteins—P-type ATPase (CadA) and transcription regulatory protein
(CadR). The CadR protein has high binding
affinity with the promoter of <i>cadR</i> and <i>cadA</i> (P
<sub><i>cadR</i></sub> and P<sub><i>cadA</i></sub>) and represses the expression of the CadA genes
downstream. However, in the presence of Cd<sup>2+</sup>, CadR will binding to Cd<sup>2+</sup> with
high specificity, thus decreases
its affinity to its promoter. So this protein acts as a strong repressor of transcription
corresponding to the change of Cd<sup>2+</sup> concentration.
</p>
<p>In our project, we constructed a gene circuit with <i>cadR</i>, P<sub><i>cadA</i></sub> and green
fluorescent protein (GFP) in <i>Escherichia coli</i>. The GFP fluorescence intensity may change
according to the Cd<sup>2+</sup> concentration,
the higher the Cd<sup>2+</sup> concentration is, the stronger the fluorescence intensity may be,
hoping that it can act as a microbial biosensor to present the intracellular cadmium concentration
which affected by different OsNramp5.
</p>
<p>The characterization experiment showed that, the <i>E.coil</i> BL21 which contained recombinant
plasmids got higher fluorescence intensity compared with non-loaded BL21 and BL21 which transformed
plasmids with GFP only. At around 2 hours,
the fluorescence intensity of engineered bacteria in 10 μM Cd<sup>2+</sup> solution was higher than
that in 1 μM Cd<sup>2+</sup> solution, since the bacteria needed some time to adapt to the
environment of Cd<sup>2+</sup>. But in general,
there was a positive correlation between Cd<sup>2+</sup> concentration and GFP fluorescence
intensity. However, the engineered bacteria was less responsive to 100 μM Cd<sup>2+</sup>
comparatively. It was probably because the engineered
bacteria exhibited lower resistance to cadmium when the concentration is too high, since the
physicochemical properties of the bacteria changed or part of the GFP protein denatured. </p>
<a id='Bioinformatics-assistedmutationsitesselection' class="target-fix"></a>
<h2>Bioinformatics-assisted mutation sites selection </h2>
<p>Directed evolution, by establishing mutation libraries and using high-throughput screening methods,
is a common strategy for protein design and modification in protein engineering, which can rapidly
improve the specific properties of the
protein. Directed evolution brings infinite possibilities for protein modification. Discontinuous
directed evolution relying on traditional methods often results in huge mutation libraries,
consuming a lot of time and energy, ending
up with bad results obtained often
<sup>[1]</sup>. In recent years, with the improvement of computer power and the optimization of
algorithms, the computer-assisted directed evolution for protein has developed quickly, which has
opened up a new direction for directed
evolution. Protein computational design based on structural simulation and energy calculation
assisting to select mutation site provides insights into "precise" directed evolution<sup>[2]</sup>.
Therefore, bioinformatics analysis can
help to reduce the redundant work for the construction of discontinuous directed evolutionary
mutation scheme. In this study, the error-prone PCR strategy was adopted to directionally evolve
OsNramp5. Due to its large error-prone mutation
library and unconcentrated mutation sites, the ideal result was not obtained. We intend to use this
method to analyze our target protein and provide theoretical reference for other researchers.
</p>
<a id='PredictionofOsNramp5proteintopologicalstructure' class="target-fix"></a>
<p><b>Prediction of OsNramp5 protein topological structure </b></p>
<p>OsNramp5 is a typical membrane protein<sup>[3]</sup>. The topological structure of a membrane protein
can be defined as its transmembrane way, the position of the membrane segment in the amino acid
sequence and the overall orientation
of the protein molecule on the membrane. The secondary structure of membrane protein can be
represented by its topological structure, which can be regarded as the intermediate of membrane
protein folding, and is the starting point
for predicting the tertiary structure of membrane protein. In order to learn more about OsNramp5
protein structure, its protein topology structure is predicted through TMHMM v2.0 (<a
href="http//www.cbs.dtu.dk/services/TMHMM/">http//www.cbs.dtu.dk/services/TMHMM/</a>).
And it turns out that OsNramp5 has 12 typical transmembrane helical regions and 76.3% of the protein
terminal are in the membrane, which is structurally similar to other Nramp families
<sup>[4]</sup>(Table S2).
</p>
<a id='PredictionofOsNramp5proteinmutationsites' class="target-fix"></a>
<p><b>Prediction of OsNramp5 protein mutation sites </b></p>
<p>Mutation hotspots of the OsNramp5 have been analyzed by searching related papers and comparing with
structure of channel proteins from the same family that have been analyzed. The sites 60D, 63N,
157G, 180E and 235M were thought to be
important for absorbing Mn2+ in OsNramp5. Different mutations of 235M result in different states of
absorbing Mn2+ and Cd2+<sup>[5,6]</sup>. The sites 60D and 63N are located outside membrane domain;
the sites 157G, 180E and 235M are
respectively located in S4, S5 and S6 transmembrane domain. "DPGN" located in the first
transmembrane domain is a conserved domain (Table S3), and is thought to be an important domain
interacting with Mn2+ homologous modeling for OsNramp5
by SWISS-MODEL<sup>[5,7-10]</sup>. The model is based on the transmembrane protein of Eremococcus
coleocola MntH (PDB Code: 6TL2.1). This figure is made by PyMOL (Fig.7). These potential mutation
sites are all from the previous reports
or software prediction. Accuracy and specific effects of these sites in OsNramp5 have not been
tested, and only taken as reference.
</p>
<div class="content_img">
<img src="./images/result10.jpg" alt="" class="result_viewer">
<p><b>Fig.7 OsNramp5 metal transporter.</b>The highly conserved "DPNG" region, located at the
c-terminus of the first transmembrane region, is consistent with the calibration of 12 cadmium
transmembrane residues in Nramp family homologue
by Gros et al. OsNramp5 homologous model was based on Eremococcus coleocola MntH (PDB Code:
6TL2.1) transmembrane protein, and its potential metal ion action region was labeled, which was
used as the preferred mutation site.</p>
</div>
<a id='Reference' class="target-fix"></a>
<h2>Reference </h2>
<p>[1] SHELDON R A, PEREIRA P C. Biocatalysis engineering: the big picture [J]. Chemical Society
Reviews, 2017, 46(10): 2678-91.</p>
<p>[2] YANG K K, WU Z, ARNOLD F H. Machine-learning-guided directed evolution for protein engineering
[J]. Nature Methods, 2019, 16(8): 687-94.</p>
<p>[3] MANI A, SANKARANARAYANAN K. In Silico Analysis of Natural Resistance-Associated Macrophage
Protein (NRAMP) Family of Transporters in Rice [J]. The Protein Journal, 2018, 37(3): 237-47.</p>
<p>[4] BOZZI A T, GAUDET R. Molecular Mechanism of Nramp-Family Transition Metal Transport [J]. J Mol
Biol, 2021, 433(16): 31.</p>
<p> [5] POTTIER M, OOMEN R, PICCO C, et al. Identification of mutations allowing Natural Resistance
Associated Macrophage Proteins (NRAMP) to discriminate against cadmium [J]. Plant Journal, 2015,
83(4): 625-37.</p>
<p>[6] JIYU L. Structure and function of Arabidopsis thaliana metal transporter NRAMPs [D]; Nanjing
Agricultural University, 2018.</p>
<p>[7] BIENERT S, WATERHOUSE A, DE BEER TJAART A P, et al. The SWISS-MODEL Repository—new features and
functionality [J]. Nucleic Acids Research, 2016, 45(D1): D313-D9.</p>
<p>[8] BERTONI M, KIEFER F, BIASINI M, et al. Modeling protein quaternary structure of homo- and
hetero-oligomers beyond binary interactions by homology [J]. Scientific Reports, 2017, 7(1): 10480.
</p>
<p>[9] STUDER G, REMPFER C, WATERHOUSE A M, et al. QMEANDisCo—distance constraints applied on model
quality estimation [J]. Bioinformatics, 2019, 36(6): 1765-71.</p>
<p>[10] STUDER G, TAURIELLO G, BIENERT S, et al. ProMod3—A versatile homology modelling toolbox [J].
PLOS Computational Biology, 2021, 17(1): e1008667.</p>
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