Difference between revisions of "Part:BBa K3089006"

 
 
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<partinfo>BBa_K3089006 short</partinfo>
  
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This part is designed for mTyr-CNK protein purification and in vitro/in vivo DOPA modification of all Mfp containing recombinant proteins. The unnatural amino acid DOPA plays an important role in Mfp’s adhesion. In natural Mfps came from posttranslational modification(PTM) in mussel foot cell, but E coli was unable to do so. Tyrosinases could oxidise tyrosine to turn it into DOPA. Although there is an existing tyrosinase in the part registry already, our mTyr-CNK has a higher efficiency in catalysing relevant reactions, thus is more suitable for DOPA modification to obtain a stronger adhesion. Instead of obtaining tyrosinase from Streptomyces sp. like in most studies, ours is found in a marine archaeon Candidatus Nitropumilus koreensis.
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This part was designed based on the paper “A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase”(Do, Kang, Yang, Cha, & Choi, 2017).
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</p>
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</html>
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<span class='h3bb'><h3>Sequence and Features</h3></span>
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<partinfo>BBa_K3089016 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K3089016 parameters</partinfo>
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<html>
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<center>
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<Figure>
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<img width="450px" src="https://2019.igem.org/wiki/images/d/d2/T--Greatbay_SCIE--P--006-figure_1.png">
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</figure>
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<figcaption>
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Figure 1. Catalytic activity of tyrosinase and advantages of mTyr-CNK.
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</figcaption>
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</center>
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<h3> Characterisation </h3>
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<p>
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mTyr-CNK is well-expressed in E. coli during our experiments, and it successfully modified all of our recombinant proteins based on Mfp5 in vitro; the co-expression system where they are expressed together inside a cell for a more efficient in vivo modification also has a very interesting potential. We believe mTyr-CNK will become an instrumental part of projects that involve DOPA modification. We also have various qualitative and quantitative data from protein expression, purification, and in vivo/ in vitro DOPA modification.
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BBa_K3089006 was characterized in the following experiments:
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</p>
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<ul>
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<li>protein expression and purification</li>
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<li> In vitro DOPA modification and NTB staining</li>
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<li> Surface coating analysis </li>
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<li> In vivo DOPA modification by co-expression</li>
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</ul>
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<h3> Protein expression </h3>
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<p>
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<i>mTyr-CNK</i> with tag for purification was cloned into pET28b and expressed in <i>E.coli</i> BL21(DE3) by 500μM IPTG for 20h at 37℃.Interestingly, after expression, the sediment of bacteria showed rose colour(Figure 2A)，probably caused by the interference of mTyr-CNK in pigment pathway of E.coli BL21(DE3) Rosetta. The exact mechanism was remained unknown since the lack of research on this tyrosinase. Results showed that obvious protein bands of mTyr-CNK(~35 kDa) could be observed on lane WC compared with lane NC (pET28b empty vector)(Figure 1B), which means the expression of this protein is well in BL21(DE3). Next, We tried to purify mTyr-CNK under native conditions, and we found bands of mTyr-CNK appeared around 35kDa on 12% SDS-PAGE gel (Figure 2B), which meant <b>it was successfully expressed and purified under native condition. Protein concentrations were measured by BCA assay, and its yield is 7mg/L. Its yield is higher than any recombinant protein in our toolbox.</b>
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</p>
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<Figure>
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<center>
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<img width="450px" src="https://2019.igem.org/wiki/images/a/a7/T--Greatbay_SCIE--Purification_of_mTyr-CNK_and_test_of_in_vitro_tyrosine_hydroxylation.png">
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</figure>
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<figcaption>
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Figure 2. Purification of mTyr-CNK and test of in vitro tyrosine hydroxylation by NBT staining. (A) Cell pellets collected after protein expression. NC: empty vector; mTyr-CNK: tyrosinase from marine microorganism; (B) SDS-PAGE of purified mTyr-CNK by affinity chromatography; (C) NBT staining to detect tyrosine hydroxylation of recombinant protein Csg-mfp5, CsgA-mfp5-mfp5, Fp151 and rBalcp19k-mfp5.
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</center>
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<br>
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<br>
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<h3> <i>In vitro</i> DOPA modification and NBT staining </h3>
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<p>
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In order to detect its modification ability on Mfp5 containing recombinant proteins, 10 ul 0.35mg/ml mTyr-CNK(in PBS, 0.02mM CuSO4）was added into 90ul protein solution of concentration 0.5m/ml（pH=6.0 PBS）for 3 hours in room temperature. Results were verified by NBT staining (<u>see details on our methods</u>). Dopa-containing proteins can be specifically stained by nitroblue tetrazolium (NBT) and glycinate solutions because they can catalyse redox-cycling reactions at an alkaline pH9. The NBT assay was thus used to confirm the successful post-translational modification of tyrosine into Dopa in modified proteins. All <i>in vitro</i> modified recombinant protein performed positive result in NBT staining test, <b>which showed tyrosines of these proteins were modified into DOPA by mTyr-CNK</b>, BSA protein was used as a negative control (Figure 2C).
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<br>
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<br>
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<h3> Surface coating analysis </h3>
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<p>
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After obtaining a small number of recombinant proteins, surface coating analysis for qualitatively assessing the surface adsorption ability of recombinant proteins was conducted on two of most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates.
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As shown in Figure3, rBalcp19k-linker-mfp5 recombinant protein showed higher surface absorption abilities on both different substrates than rBalcp19k without fusion of mfp5 on its C-terminal.
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It’s suggested that Mfp improves the coating ability of rBalcp19k-linker-mfp5 fusion proteins. The In-vitro DOPA modification by mTyr-CNK tyrosinase significantly improved its surface absorption abilities, which suggested the positive contribution of DOPA in adhesive protein performances.
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</p>
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 +
<Figure>
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<center>
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<img width="300px" src="https://2019.igem.org/wiki/images/3/39/T--Greatbay_SCIE--surface_coating_analysis_2.jpeg">
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</figure>
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<figcaption>
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Figure 3.NBT staining to detect tyrosine hydroxylation of recombinant protein Csg-mfp5, CsgA-mfp5-mfp5, Fp151 and rBalcp19k-mfp5.
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</figcaption>.
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</center>
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<Figure>
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<img width="600px" src="https://static.igem.org/mediawiki/parts/8/8e/T--Greatbay_SCIE--P--Surface_coating.jpeg">
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</figure>
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<figcaption>
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Figure 4. Surface coating analysis assay.
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</figcaption>
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<p>
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As shown in Figure 4, Mfp5 related proteins (unmodified) exhibited higher surface absorption abilities than other recombinant proteins, whereas almost all absorbed BSA were washed away. This revealed the functional advantage of Mfp5 since we could combine it with different proteins and predict the properties of recombinant proteins, making them more adaptable to diverse conditions. Furthermore, <b>the DOPA modification by mTyr-CNK significantly improved the surface absorption abilities of Mfp-related recombinant proteins</b>, which suggested the positive contribution of DOPA in adhesive protein performances. Future research on the mechanism of DOPA may suggest more applications of DOPA; it is possible to even integrate DOPA into other proteins like fibrin to enhance them.
 +
 +
<figcaption>
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Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).
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</figcaption>
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<h3><i>In vivo</i> DOPA modification by co-expression</h3>
 +
<p>
 +
Other researches suggested in vivo DOPA modification could modify a higher proportion of tyrosines to DOPA and simplify later processing. We may be able to obtain better-modified proteins in an easier way. Thus we created a prototype in vivo co-expression bi-plasmid system of recombinant proteins and mTyr-CNK. Because the pET vector can be used in combination with a vector system with p15A replicon. The recombinant pACYC-mTyr-CNK plasmid was prepared from pACYCDuet-1, which carries the p15A origin. We transformed the two-plasmid system into BL21(DE3) to try in-vivo modification (Figure 4A)。We used the same methods in the induction of mTyr-CNK in Figure 2 and protein purification as their non-co-expression versions to obtain co-CsgA-mfp5, co-CsgA-mfp5-mfp5, co-Fp151, co-rBalcp19k-mfp5(Figure 4BCD). Their final yield were 0.4mg/L, 0.4mg/L, 4.75mg/L, 3.25mg/L, respectively. We used NBT staining to verify the presence of DOPA. Unfortunately, the test gave a negative result.
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</p>
 +
 +
<Figure>
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<img width="600px" src="https://2019.igem.org/wiki/images/3/3c/T--Greatbay_SCIE--Co-expression_and_test.png">
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</figure>
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<figcaption>
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Figure 5. Co-expression and test of in vivo tyrosine hydroxylation by NBT staining. Purple colour indicates the presence of DOPA; L-DOPA was used as a positive control.
 +
</figcaption>
 +
 +
<p>
 +
mTyr-CNK didn’t oxidise tyrosine into DOPA, which was likely caused by low expression of tyrosinase mTyr-CNK in the co-expression system. As shown in Figure 7, mTyr-CNK expressed at a high level when expressed alone, and didn’t express in co-expression strains under the same expression conditions. Recombinant proteins expression also fell, based on this issue, we modelled the co-expression system to discuss the mechanism behind it. We expected modelling could suggest better gene circuits to improve the DOPA modification function of the co-expression system.
 +
</p>
 +
 +
<Figure>
 +
<img width="600px" src="https://2019.igem.org/wiki/images/5/54/T--Greatbay_SCIE--SDS-PAGE_of_whole_cell_proteins.png">
 +
</figure>
 +
 +
<figcaption>
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Figure 6. SDS-PAGE of whole-cell proteins of co-expression strains. Induced expression of mTyr-CNK. (B) SDS-PAGE of co-expression recombinant proteins. The black arrow represents the predicted position of mTyr-CNK and red arrows mark the predicted position of recombinant proteins.
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</figcaption>
 +
 +
<h3>Improve the yield of mTyr-CNK</h3>
 +
 +
<p><b>Characterized by iGEM2020_Beijing_4ELEVEN</b></p>
 +
We have improved the previous part BBa_K3089006 by using a series of new RBSs that differ in their expression efficiencies in expressing tyrosinase mTyr-CNK.</p>
 +
 +
<p>We began with randomly arranging six pairs in the system's RBS sequence TAAGTATAAGNNNNNNATAT, and verifying the resulting RBSs' strengths with RBS calculator. 15 of the strongest RBSs from verification are taken into our RBS library. </p>
 +
 +
<center>
 +
<Figure>
 +
<img width="450px" src="https://2020.igem.org/wiki/images/2/2e/T--BEIJING_4ELEVEN--Improvement_Figure_1.png">
 +
</figure>
 +
 +
<figcaption>
 +
Figure 1. Overall design of our RBS library.
 +
</figcaption>
 +
</center>
 +
 +
 +
<p>These RBSs are then integrated into the expression system of mTyr-CNK through Goldengate, transformed into E. coli BL21 along with the original part BBa_K3089006 as control group. </p>
 +
 +
<p>All the strains were cultivated in LB medium with 0.5mM IPTG, 25°C, for 20 hours. 100 mL culture were collected for proteins purification. Then the products are verified by SDS-PAGE and quantified through BCA assay.</p>
 +
 +
<center>
 +
<Figure>
 +
<img width="450px" src="https://2020.igem.org/wiki/images/2/21/T--BEIJING_4ELEVEN--Improvement_Figure_2.png">
 +
</figure>
 +
 +
<figcaption>
 +
Figure 2. SDS-PAGE gel of our RBS library.
 +
</figcaption>
 +
</center>
 +
 +
<p>In the results, we have succeeded in improving the production of mTyr-CNK. From the SDS-PAGE, we can clearly identify correct bands of mTyr-CNK (Figure 2). <b>In BCA assay results , 6 of the 15 RBSs have improved the tyrosinase yield, the highest one is about 4 times to the control (Figure 3).<b/></p>
 +
 +
 +
<center>
 +
<Figure>
 +
<img width="450px" src="https://2020.igem.org/wiki/images/c/c4/T--BEIJING_4ELEVEN--Improvement_Figure_3_BCA.png">
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</figure>
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<figcaption>
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Figure 3. BCA assay of our RBS library
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</figcaption>
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</center>

Latest revision as of 03:22, 28 October 2020

mTyr-CNK tyrosinase


This part is designed for mTyr-CNK protein purification and in vitro/in vivo DOPA modification of all Mfp containing recombinant proteins. The unnatural amino acid DOPA plays an important role in Mfp’s adhesion. In natural Mfps came from posttranslational modification(PTM) in mussel foot cell, but E coli was unable to do so. Tyrosinases could oxidise tyrosine to turn it into DOPA. Although there is an existing tyrosinase in the part registry already, our mTyr-CNK has a higher efficiency in catalysing relevant reactions, thus is more suitable for DOPA modification to obtain a stronger adhesion. Instead of obtaining tyrosinase from Streptomyces sp. like in most studies, ours is found in a marine archaeon Candidatus Nitropumilus koreensis. This part was designed based on the paper “A tyrosinase, mTyr-CNK, that is functionally available as a monophenol monooxygenase”(Do, Kang, Yang, Cha, & Choi, 2017).

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 274
    Illegal XhoI site found at 244
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 274
    Illegal PstI site found at 23
  • 1000
    COMPATIBLE WITH RFC[1000]


Figure 1. Catalytic activity of tyrosinase and advantages of mTyr-CNK.

Characterisation

mTyr-CNK is well-expressed in E. coli during our experiments, and it successfully modified all of our recombinant proteins based on Mfp5 in vitro; the co-expression system where they are expressed together inside a cell for a more efficient in vivo modification also has a very interesting potential. We believe mTyr-CNK will become an instrumental part of projects that involve DOPA modification. We also have various qualitative and quantitative data from protein expression, purification, and in vivo/ in vitro DOPA modification. BBa_K3089006 was characterized in the following experiments:

  • protein expression and purification
  • In vitro DOPA modification and NTB staining
  • Surface coating analysis
  • In vivo DOPA modification by co-expression

Protein expression

mTyr-CNK with tag for purification was cloned into pET28b and expressed in E.coli BL21(DE3) by 500μM IPTG for 20h at 37℃.Interestingly, after expression, the sediment of bacteria showed rose colour(Figure 2A)，probably caused by the interference of mTyr-CNK in pigment pathway of E.coli BL21(DE3) Rosetta. The exact mechanism was remained unknown since the lack of research on this tyrosinase. Results showed that obvious protein bands of mTyr-CNK(~35 kDa) could be observed on lane WC compared with lane NC (pET28b empty vector)(Figure 1B), which means the expression of this protein is well in BL21(DE3). Next, We tried to purify mTyr-CNK under native conditions, and we found bands of mTyr-CNK appeared around 35kDa on 12% SDS-PAGE gel (Figure 2B), which meant it was successfully expressed and purified under native condition. Protein concentrations were measured by BCA assay, and its yield is 7mg/L. Its yield is higher than any recombinant protein in our toolbox.

Figure 2. Purification of mTyr-CNK and test of in vitro tyrosine hydroxylation by NBT staining. (A) Cell pellets collected after protein expression. NC: empty vector; mTyr-CNK: tyrosinase from marine microorganism; (B) SDS-PAGE of purified mTyr-CNK by affinity chromatography; (C) NBT staining to detect tyrosine hydroxylation of recombinant protein Csg-mfp5, CsgA-mfp5-mfp5, Fp151 and rBalcp19k-mfp5.

In vitro DOPA modification and NBT staining

In order to detect its modification ability on Mfp5 containing recombinant proteins, 10 ul 0.35mg/ml mTyr-CNK(in PBS, 0.02mM CuSO4）was added into 90ul protein solution of concentration 0.5m/ml（pH=6.0 PBS）for 3 hours in room temperature. Results were verified by NBT staining (see details on our methods). Dopa-containing proteins can be specifically stained by nitroblue tetrazolium (NBT) and glycinate solutions because they can catalyse redox-cycling reactions at an alkaline pH9. The NBT assay was thus used to confirm the successful post-translational modification of tyrosine into Dopa in modified proteins. All in vitro modified recombinant protein performed positive result in NBT staining test, which showed tyrosines of these proteins were modified into DOPA by mTyr-CNK, BSA protein was used as a negative control (Figure 2C).

Surface coating analysis

After obtaining a small number of recombinant proteins, surface coating analysis for qualitatively assessing the surface adsorption ability of recombinant proteins was conducted on two of most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates. As shown in Figure3, rBalcp19k-linker-mfp5 recombinant protein showed higher surface absorption abilities on both different substrates than rBalcp19k without fusion of mfp5 on its C-terminal. It’s suggested that Mfp improves the coating ability of rBalcp19k-linker-mfp5 fusion proteins. The In-vitro DOPA modification by mTyr-CNK tyrosinase significantly improved its surface absorption abilities, which suggested the positive contribution of DOPA in adhesive protein performances.

Figure 3.NBT staining to detect tyrosine hydroxylation of recombinant protein Csg-mfp5, CsgA-mfp5-mfp5, Fp151 and rBalcp19k-mfp5.
.
Figure 4. Surface coating analysis assay.

As shown in Figure 4, Mfp5 related proteins (unmodified) exhibited higher surface absorption abilities than other recombinant proteins, whereas almost all absorbed BSA were washed away. This revealed the functional advantage of Mfp5 since we could combine it with different proteins and predict the properties of recombinant proteins, making them more adaptable to diverse conditions. Furthermore, the DOPA modification by mTyr-CNK significantly improved the surface absorption abilities of Mfp-related recombinant proteins, which suggested the positive contribution of DOPA in adhesive protein performances. Future research on the mechanism of DOPA may suggest more applications of DOPA; it is possible to even integrate DOPA into other proteins like fibrin to enhance them.

Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).

In vivo DOPA modification by co-expression

Other researches suggested in vivo DOPA modification could modify a higher proportion of tyrosines to DOPA and simplify later processing. We may be able to obtain better-modified proteins in an easier way. Thus we created a prototype in vivo co-expression bi-plasmid system of recombinant proteins and mTyr-CNK. Because the pET vector can be used in combination with a vector system with p15A replicon. The recombinant pACYC-mTyr-CNK plasmid was prepared from pACYCDuet-1, which carries the p15A origin. We transformed the two-plasmid system into BL21(DE3) to try in-vivo modification (Figure 4A)。We used the same methods in the induction of mTyr-CNK in Figure 2 and protein purification as their non-co-expression versions to obtain co-CsgA-mfp5, co-CsgA-mfp5-mfp5, co-Fp151, co-rBalcp19k-mfp5(Figure 4BCD). Their final yield were 0.4mg/L, 0.4mg/L, 4.75mg/L, 3.25mg/L, respectively. We used NBT staining to verify the presence of DOPA. Unfortunately, the test gave a negative result.

Figure 5. Co-expression and test of in vivo tyrosine hydroxylation by NBT staining. Purple colour indicates the presence of DOPA; L-DOPA was used as a positive control.

mTyr-CNK didn’t oxidise tyrosine into DOPA, which was likely caused by low expression of tyrosinase mTyr-CNK in the co-expression system. As shown in Figure 7, mTyr-CNK expressed at a high level when expressed alone, and didn’t express in co-expression strains under the same expression conditions. Recombinant proteins expression also fell, based on this issue, we modelled the co-expression system to discuss the mechanism behind it. We expected modelling could suggest better gene circuits to improve the DOPA modification function of the co-expression system.

Figure 6. SDS-PAGE of whole-cell proteins of co-expression strains. Induced expression of mTyr-CNK. (B) SDS-PAGE of co-expression recombinant proteins. The black arrow represents the predicted position of mTyr-CNK and red arrows mark the predicted position of recombinant proteins.

Improve the yield of mTyr-CNK

Characterized by iGEM2020_Beijing_4ELEVEN

We have improved the previous part BBa_K3089006 by using a series of new RBSs that differ in their expression efficiencies in expressing tyrosinase mTyr-CNK.

We began with randomly arranging six pairs in the system's RBS sequence TAAGTATAAGNNNNNNATAT, and verifying the resulting RBSs' strengths with RBS calculator. 15 of the strongest RBSs from verification are taken into our RBS library.

Figure 1. Overall design of our RBS library.

These RBSs are then integrated into the expression system of mTyr-CNK through Goldengate, transformed into E. coli BL21 along with the original part BBa_K3089006 as control group.

All the strains were cultivated in LB medium with 0.5mM IPTG, 25°C, for 20 hours. 100 mL culture were collected for proteins purification. Then the products are verified by SDS-PAGE and quantified through BCA assay.

Figure 2. SDS-PAGE gel of our RBS library.

In the results, we have succeeded in improving the production of mTyr-CNK. From the SDS-PAGE, we can clearly identify correct bands of mTyr-CNK (Figure 2). In BCA assay results , 6 of the 15 RBSs have improved the tyrosinase yield, the highest one is about 4 times to the control (Figure 3).

Figure 3. BCA assay of our RBS library