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Research article
Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA
Department of Biomolecular and Chemical Engineering, and Department of Nursing, Dongyang University, Yeongju 36040, Republic of Korea
Correspondence to:J. Microbiol. Biotechnol. 2020; 30(7): 1097-1103
Published July 28, 2020 https://doi.org/10.4014/jmb.2001.01053
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Graphical Abstract
Introduction
Bacterial surface display is a protein engineering technique used for display of a target, such as a peptide or protein (enzyme) on the surface of bacteria. Bacterial cell surface display systems have been employed for various biotechnological and industrial applications, such as whole-cell biocatalysts, biosensors, bioabsorbents and affinity-based screening, antibody epitope mapping, and vaccine delivery [1-3]. In particular,
However, the
In this study, we developed an efficient
Materials and Methods
Bacterial Strains and Culture Conditions
Table 1 shows all bacterial strains and plasmids employed in this study.
-
Table 1 . The bacterial strains and plasmids used in this study.
Strain or Plasmid Relevant Characteristics Reference or Source E. coli strainsXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´proAB lacI qZ ΔM15 Tn10 (Tetr)]Stratagenea XL10-Gold Tetr ∆( mcrA )183 ∆(mcrCB-hsdSMR-mrr )173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´proAB lacI qZ ∆M15 Tn10 (Tetr) Amy Camr]Stratagenea Plasmids pTrc99A 4.2 kb; Apr, trc promoterPharmaciab pTrcM 4.9 kb; pTrc99A derivative containing full-length 747 bp fragment of E. coli mipA This study pTrcMV140 4.6 kb; pTrc99A derivative containing 420 bp fragment of E. coli mipA This study pTrcMV176 4.7 kb; pTrc99A derivative containing 528 bp fragment of E. coli mipA This study pTrcMK179 4.7 kb; pTrc99A derivative containing 537 bp fragment of E. coli mipA This study pTrcMV226 4.9 kb; pTrc99A derivative containing 678 bp fragment of E. coli mipA This study pTrcMV232 4.9 kb; pTrc99A derivative containing 696 bp fragment of E. coli mipA This study pTrcMK234 4.9 kb; pTrc99A derivative containing 702 bp fragment of E. coli mipA This study pTrcMV140PL 6.0 kb; pTrcMV140 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV176PL 6.1 kb; pTrcMV176 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMK179PL 6.1 kb; pTrcMV179 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV226PL 6.3 kb; pTrcMV226 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV232PL 6.3 kb; pTrcMV232 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMK234PL 6.3 kb; pTrcMK234 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV140BA 5.9 kb; pTrcMV140 derivative containing B. subtilis α-amylase gene fused with FLAG-tagThis study a Stratagene Cloning System, USA.
b Pharmacia Biotech, Uppsala, Sweden.
DNA Manipulation
Table 2 shows the primers used in this study. Polymerase chain reaction (PCR) to amplify target genes described in Table 2 was performed with a PCR Thermal Cycler MP (Takara Shuzo Co., Ltd., Japan) using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Germany). DNA sequencing was carried out using the BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer Co., USA) with
-
Table 2 . The list of primers used in the PCR experiments.
Primer no. Sequencea Gene to be amplified Template 1 5-g gaattc ATGACCAAACTCAAACTTCTGGCAFull-length mipA geneE. coli W3110 chromosome2 5-gc tctaga TCAGAATTTGTAGGTGATCCCGGT3 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val140 positionE. coli W3110 chromosome4 5-gc tctaga GACGATGCCGTTGCTGTTATCC5 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val176 positionE. coli W3110 chromosome6 5-gc tctaga TACGCCATAATAGTATTCGTTCTG7 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Lys179 positionE. coli W3110 chromosome8 5-gc tctaga TTTGCGCGATACGCCATAATAGTA9 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val226 positionE. coli W3110 chromosome10 5-gc tctaga AACTTCATCAGACAGACGGGTGTA11 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val232 positionE. coli W3110 chromosome12 5-gc tctaga CACCATCGGGCTGTCAGTAACTTC13 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Lys234 positionE. coli W3110 chromosome14 5-gc tctaga TTTATCCACCATCGGGCTGTCAGT15 5-gc tctaga ATGGGTGTATTTGACTACAAGAACP. fluorescens SIK W1 lipase gene fused with FLAG tagP. fluorescens SIK W1 chromosome [28,29]16 5-ccc aagctt ttacttgtcgtcatcgtccttgtagtcACTGATCAGCACACCa Restriction enzyme sites are show
tliA , which encodes a thermostable lipase (476 aa; 49.9 kDa).
SDS-PAGE and Immunoblotting
To confirm an enzyme display on the
Immunofluorescence Microscopy
For fluorescence imaging, cells induced with 1 mM IPTG for 4 h were harvested by centrifugation for 5 min at 3,500 ×
Measurement of Enzymatic Activities
The activity of the
The activity of
In this formula, ODSample, ODSTD, and ODBuffer are the optical density values of the sample, the 400 μM glucose standard and assay buffer, respectively, t is the incubation time, and n is the dilution factor. One unit (U) was defined as the amount of enzyme required to produce 1 μmol of glucose per minute under the assay conditions.
All activity assays were independently performed in triplicate, and the standard deviations were determined.
Results and Discussion
Design of the E. coli Surface Display System Using an mipA Gene
The function of
The outer membrane topology of MipA was first predicted using PRED-TMBB (http://bioinformatics.biol.uoa.gr/PRED-TMBB/ ) [27]. As shown in Fig. 1A, the MipA protein contains five extracellular loops that form a β- sheet protruding from the cell surface. Among these loops, the third, fourth and fifth loops were primarily considered, since they likely have stronger and more stable anchoring locations in the β-barrel structure of
-
Fig. 1.
Construction of the MipA-fused display system. (A ) The structure of MipA in theE. coli outer membrane. This topology was an arbitrary redrawing of the image predicted by PRED-TMBB [27]. Each gray circle indicates the fusion position of MipA with lipase: V140 in the third extracellular loop; V176 and K179 in the fourth extracellular loop; and V226, V232, and K234 in the fifth extracellular loop. (B) A schematic diagram of the expression system for MipA-fused target genes.
Construction of the Lipase Display System on the E. coli Surface
For construction of expression systems composed of truncated MipA fused to a target protein (Fig. 1B), the full- length
To display a lipase on the
Lipase Activity on the E. coli Surface
To test which anchoring motif of the
-
Fig. 2.
Comparison of lipase activity between the MipA anchoring motifs developed in this study and the previously reported motifs FadL [8], OprF [9], and YiaT [20]. All activity assays were independently performed in triplicate, and the standard deviations were determined. One unit (U) of lipase activity was defined as the amount of enzyme that releases 1 μmol ofE. coli XL10-Gold (pTrcM) is indicated as a control.p -nitrophenol per minute.
Confirmation of Lipase Display on the E. coli Surface
To confirm that localization of lipase was displayed on the surface using MV140 as an anchoring motif, the total lysate, outer membrane proteins, and soluble protein fractions of the
-
Fig. 3.
SDS-PAGE and western blotting analyses for total, outer membrane, and soluble fractions from First lane in each gel indicates the molecular weight size markers (kDa). T, whole cell lysate; M, outer membrane protein fractions; and S, soluble proteins. Arrows indicate the MV140-fused lipase.E. coli XL10-Gold harboring pTrcM (control) (A) and pTrcMV140PL (B).
Confocal Microscopic Analysis of Lipase Display on the E. coli Surface
Additionally, the localization of lipase was confirmed by confocal microscopy. After cultivation, the cells were labeled with FITC-conjugated anti-FLAG antibody probe, which can recognize the FLAG tag linked to the C- terminus of lipase.
-
Fig. 4.
Differential interference microscope images (upper) and confocal immunofluorescence microscope images (lower) of Each scale bar represents 5 μm.E. coli XL10-Gold cells harboring pTrcM (control; left images) and pTrcMV140PL (right images).
Display of α-Amylase on the E. coli Surface Using the MV140 as an Anchoring Motif
To demonstrate the general use of MV140 motif,
-
Fig. 5.
Display of (B. subtilis α-amylase on theE. coli cell surface.A ) Western blotting analyses of the outer membrane fractions ofE. coli XL10-Gold cells harboring pTrcM (control) or pTrcMV140BA. M indicates the molecular weight size markers (kDa). (B ) The α-amylase activity ofE. coli XL10-Gold cells harboring pTrcM (control), pTrcMV140BA or pTrcYiaTR232BA [20].
Next, we examined whether the displayed α-amylase proteins were active. The specific whole-cell α-amylase activities of the recombinant
In summary, a new cell surface display system was developed using the
Acknowledgments
This study was supported by a grant from Dong Yang University in 2018. The author gratefully acknowledges the technical assistance provided by Hoon Jae Lee.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
References
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Related articles in JMB

Article
Research article
J. Microbiol. Biotechnol. 2020; 30(7): 1097-1103
Published online July 28, 2020 https://doi.org/10.4014/jmb.2001.01053
Copyright © The Korean Society for Microbiology and Biotechnology.
Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA
Mee Jung Han*
Department of Biomolecular and Chemical Engineering, and Department of Nursing, Dongyang University, Yeongju 36040, Republic of Korea
Correspondence to:Mee Jung Han
mjhan75@dyu.ac.kr
Abstract
Bacterial surface display systems have been developed for various applications in biotechnology and industry. Particularly, the discovery and design of anchoring motifs is highly important for the successful display of a target protein or peptide on the surface of bacteria. In this study, an efficient display system on Escherichia coli was developed using novel anchoring motifs designed from the E. coli mipA gene. Using the C-terminal fusion system of an industrial enzyme, Pseudomonas fluorescens lipase, six possible fusion sites, V140, V176, K179, V226, V232, and K234, which were truncated from the C-terminal end of the mipA gene (MV140, MV176, MV179, MV226, MV232, and MV234) were examined. The whole-cell lipase activities showed that MV140 was the best among the six anchoring motifs. Furthermore, the lipase activity obtained using MV140 as the anchoring motif was approximately 20-fold higher than that of the previous anchoring motifs FadL and OprF but slightly higher than that of YiaTR232. Western blotting and confocal microscopy further confirmed the localization of the fusion lipase displayed on the E. coli surface using the truncated MV140. Additionally the MV140 motif could be used for successfully displaying another industrial enzyme, α-amylase from Bacillus subtilis. These results showed that the fusion proteins using the MV140 motif had notably high enzyme activities and did not exert any adverse effects on either cell growth or outer membrane integrity. Thus, this study shows that MipA can be used as a novel anchoring motif for more efficient bacterial surface display in the biotechnological and industrial fields.
Keywords: E. coli MipA, cell surface display, outer membrane protein
Introduction
Bacterial surface display is a protein engineering technique used for display of a target, such as a peptide or protein (enzyme) on the surface of bacteria. Bacterial cell surface display systems have been employed for various biotechnological and industrial applications, such as whole-cell biocatalysts, biosensors, bioabsorbents and affinity-based screening, antibody epitope mapping, and vaccine delivery [1-3]. In particular,
However, the
In this study, we developed an efficient
Materials and Methods
Bacterial Strains and Culture Conditions
Table 1 shows all bacterial strains and plasmids employed in this study.
-
Table 1 . The bacterial strains and plasmids used in this study..
Strain or Plasmid Relevant Characteristics Reference or Source E. coli strainsXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´proAB lacI qZ ΔM15 Tn10 (Tetr)]Stratagenea XL10-Gold Tetr ∆( mcrA )183 ∆(mcrCB-hsdSMR-mrr )173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´proAB lacI qZ ∆M15 Tn10 (Tetr) Amy Camr]Stratagenea Plasmids pTrc99A 4.2 kb; Apr, trc promoterPharmaciab pTrcM 4.9 kb; pTrc99A derivative containing full-length 747 bp fragment of E. coli mipA This study pTrcMV140 4.6 kb; pTrc99A derivative containing 420 bp fragment of E. coli mipA This study pTrcMV176 4.7 kb; pTrc99A derivative containing 528 bp fragment of E. coli mipA This study pTrcMK179 4.7 kb; pTrc99A derivative containing 537 bp fragment of E. coli mipA This study pTrcMV226 4.9 kb; pTrc99A derivative containing 678 bp fragment of E. coli mipA This study pTrcMV232 4.9 kb; pTrc99A derivative containing 696 bp fragment of E. coli mipA This study pTrcMK234 4.9 kb; pTrc99A derivative containing 702 bp fragment of E. coli mipA This study pTrcMV140PL 6.0 kb; pTrcMV140 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV176PL 6.1 kb; pTrcMV176 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMK179PL 6.1 kb; pTrcMV179 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV226PL 6.3 kb; pTrcMV226 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV232PL 6.3 kb; pTrcMV232 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMK234PL 6.3 kb; pTrcMK234 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV140BA 5.9 kb; pTrcMV140 derivative containing B. subtilis α-amylase gene fused with FLAG-tagThis study a Stratagene Cloning System, USA..
b Pharmacia Biotech, Uppsala, Sweden..
DNA Manipulation
Table 2 shows the primers used in this study. Polymerase chain reaction (PCR) to amplify target genes described in Table 2 was performed with a PCR Thermal Cycler MP (Takara Shuzo Co., Ltd., Japan) using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Germany). DNA sequencing was carried out using the BigDye Terminator Cycle Sequencing Kit (Perkin-Elmer Co., USA) with
-
Table 2 . The list of primers used in the PCR experiments..
Primer no. Sequencea Gene to be amplified Template 1 5-g gaattc ATGACCAAACTCAAACTTCTGGCAFull-length mipA geneE. coli W3110 chromosome2 5-gc tctaga TCAGAATTTGTAGGTGATCCCGGT3 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val140 positionE. coli W3110 chromosome4 5-gc tctaga GACGATGCCGTTGCTGTTATCC5 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val176 positionE. coli W3110 chromosome6 5-gc tctaga TACGCCATAATAGTATTCGTTCTG7 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Lys179 positionE. coli W3110 chromosome8 5-gc tctaga TTTGCGCGATACGCCATAATAGTA9 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val226 positionE. coli W3110 chromosome10 5-gc tctaga AACTTCATCAGACAGACGGGTGTA11 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val232 positionE. coli W3110 chromosome12 5-gc tctaga CACCATCGGGCTGTCAGTAACTTC13 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Lys234 positionE. coli W3110 chromosome14 5-gc tctaga TTTATCCACCATCGGGCTGTCAGT15 5-gc tctaga ATGGGTGTATTTGACTACAAGAACP. fluorescens SIK W1 lipase gene fused with FLAG tagP. fluorescens SIK W1 chromosome [28,29]16 5-ccc aagctt ttacttgtcgtcatcgtccttgtagtcACTGATCAGCACACCa Restriction enzyme sites are show.
tliA , which encodes a thermostable lipase (476 aa; 49.9 kDa)..
SDS-PAGE and Immunoblotting
To confirm an enzyme display on the
Immunofluorescence Microscopy
For fluorescence imaging, cells induced with 1 mM IPTG for 4 h were harvested by centrifugation for 5 min at 3,500 ×
Measurement of Enzymatic Activities
The activity of the
The activity of
In this formula, ODSample, ODSTD, and ODBuffer are the optical density values of the sample, the 400 μM glucose standard and assay buffer, respectively, t is the incubation time, and n is the dilution factor. One unit (U) was defined as the amount of enzyme required to produce 1 μmol of glucose per minute under the assay conditions.
All activity assays were independently performed in triplicate, and the standard deviations were determined.
Results and Discussion
Design of the E. coli Surface Display System Using an mipA Gene
The function of
The outer membrane topology of MipA was first predicted using PRED-TMBB (http://bioinformatics.biol.uoa.gr/PRED-TMBB/ ) [27]. As shown in Fig. 1A, the MipA protein contains five extracellular loops that form a β- sheet protruding from the cell surface. Among these loops, the third, fourth and fifth loops were primarily considered, since they likely have stronger and more stable anchoring locations in the β-barrel structure of
-
Figure 1.
Construction of the MipA-fused display system. (A ) The structure of MipA in theE. coli outer membrane. This topology was an arbitrary redrawing of the image predicted by PRED-TMBB [27]. Each gray circle indicates the fusion position of MipA with lipase: V140 in the third extracellular loop; V176 and K179 in the fourth extracellular loop; and V226, V232, and K234 in the fifth extracellular loop. (B) A schematic diagram of the expression system for MipA-fused target genes.
Construction of the Lipase Display System on the E. coli Surface
For construction of expression systems composed of truncated MipA fused to a target protein (Fig. 1B), the full- length
To display a lipase on the
Lipase Activity on the E. coli Surface
To test which anchoring motif of the
-
Figure 2.
Comparison of lipase activity between the MipA anchoring motifs developed in this study and the previously reported motifs FadL [8], OprF [9], and YiaT [20]. All activity assays were independently performed in triplicate, and the standard deviations were determined. One unit (U) of lipase activity was defined as the amount of enzyme that releases 1 μmol ofE. coli XL10-Gold (pTrcM) is indicated as a control.p -nitrophenol per minute.
Confirmation of Lipase Display on the E. coli Surface
To confirm that localization of lipase was displayed on the surface using MV140 as an anchoring motif, the total lysate, outer membrane proteins, and soluble protein fractions of the
-
Figure 3.
SDS-PAGE and western blotting analyses for total, outer membrane, and soluble fractions from First lane in each gel indicates the molecular weight size markers (kDa). T, whole cell lysate; M, outer membrane protein fractions; and S, soluble proteins. Arrows indicate the MV140-fused lipase.E. coli XL10-Gold harboring pTrcM (control) (A) and pTrcMV140PL (B).
Confocal Microscopic Analysis of Lipase Display on the E. coli Surface
Additionally, the localization of lipase was confirmed by confocal microscopy. After cultivation, the cells were labeled with FITC-conjugated anti-FLAG antibody probe, which can recognize the FLAG tag linked to the C- terminus of lipase.
-
Figure 4.
Differential interference microscope images (upper) and confocal immunofluorescence microscope images (lower) of Each scale bar represents 5 μm.E. coli XL10-Gold cells harboring pTrcM (control; left images) and pTrcMV140PL (right images).
Display of α-Amylase on the E. coli Surface Using the MV140 as an Anchoring Motif
To demonstrate the general use of MV140 motif,
-
Figure 5.
Display of (B. subtilis α-amylase on theE. coli cell surface.A ) Western blotting analyses of the outer membrane fractions ofE. coli XL10-Gold cells harboring pTrcM (control) or pTrcMV140BA. M indicates the molecular weight size markers (kDa). (B ) The α-amylase activity ofE. coli XL10-Gold cells harboring pTrcM (control), pTrcMV140BA or pTrcYiaTR232BA [20].
Next, we examined whether the displayed α-amylase proteins were active. The specific whole-cell α-amylase activities of the recombinant
In summary, a new cell surface display system was developed using the
Acknowledgments
This study was supported by a grant from Dong Yang University in 2018. The author gratefully acknowledges the technical assistance provided by Hoon Jae Lee.
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

Fig 2.

Fig 3.

Fig 4.

Fig 5.

-
Table 1 . The bacterial strains and plasmids used in this study..
Strain or Plasmid Relevant Characteristics Reference or Source E. coli strainsXL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F´proAB lacI qZ ΔM15 Tn10 (Tetr)]Stratagenea XL10-Gold Tetr ∆( mcrA )183 ∆(mcrCB-hsdSMR-mrr )173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac Hte [F´proAB lacI qZ ∆M15 Tn10 (Tetr) Amy Camr]Stratagenea Plasmids pTrc99A 4.2 kb; Apr, trc promoterPharmaciab pTrcM 4.9 kb; pTrc99A derivative containing full-length 747 bp fragment of E. coli mipA This study pTrcMV140 4.6 kb; pTrc99A derivative containing 420 bp fragment of E. coli mipA This study pTrcMV176 4.7 kb; pTrc99A derivative containing 528 bp fragment of E. coli mipA This study pTrcMK179 4.7 kb; pTrc99A derivative containing 537 bp fragment of E. coli mipA This study pTrcMV226 4.9 kb; pTrc99A derivative containing 678 bp fragment of E. coli mipA This study pTrcMV232 4.9 kb; pTrc99A derivative containing 696 bp fragment of E. coli mipA This study pTrcMK234 4.9 kb; pTrc99A derivative containing 702 bp fragment of E. coli mipA This study pTrcMV140PL 6.0 kb; pTrcMV140 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV176PL 6.1 kb; pTrcMV176 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMK179PL 6.1 kb; pTrcMV179 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV226PL 6.3 kb; pTrcMV226 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV232PL 6.3 kb; pTrcMV232 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMK234PL 6.3 kb; pTrcMK234 derivative containing P. fluorescens SIK W1 lipase gene fused with FLAG-tagThis study pTrcMV140BA 5.9 kb; pTrcMV140 derivative containing B. subtilis α-amylase gene fused with FLAG-tagThis study a Stratagene Cloning System, USA..
b Pharmacia Biotech, Uppsala, Sweden..
-
Table 2 . The list of primers used in the PCR experiments..
Primer no. Sequencea Gene to be amplified Template 1 5-g gaattc ATGACCAAACTCAAACTTCTGGCAFull-length mipA geneE. coli W3110 chromosome2 5-gc tctaga TCAGAATTTGTAGGTGATCCCGGT3 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val140 positionE. coli W3110 chromosome4 5-gc tctaga GACGATGCCGTTGCTGTTATCC5 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val176 positionE. coli W3110 chromosome6 5-gc tctaga TACGCCATAATAGTATTCGTTCTG7 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Lys179 positionE. coli W3110 chromosome8 5-gc tctaga TTTGCGCGATACGCCATAATAGTA9 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val226 positionE. coli W3110 chromosome10 5-gc tctaga AACTTCATCAGACAGACGGGTGTA11 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Val232 positionE. coli W3110 chromosome12 5-gc tctaga CACCATCGGGCTGTCAGTAACTTC13 5-g gaattc ATGACCAAACTCAAACTTCTGGCATruncated mipA at Lys234 positionE. coli W3110 chromosome14 5-gc tctaga TTTATCCACCATCGGGCTGTCAGT15 5-gc tctaga ATGGGTGTATTTGACTACAAGAACP. fluorescens SIK W1 lipase gene fused with FLAG tagP. fluorescens SIK W1 chromosome [28,29]16 5-ccc aagctt ttacttgtcgtcatcgtccttgtagtcACTGATCAGCACACCa Restriction enzyme sites are show.
tliA , which encodes a thermostable lipase (476 aa; 49.9 kDa)..
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