Difference between revisions of "Part:BBa K2235009"
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==Introduction==
 
==Introduction==
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This biobrick is a constitute of T7 promoter and RBS followed by the sialidase enzyme coding site. Sialidase enzyme has the potential to digest terminal sialic acids in a glycoprotein. The sequence originates from the species ''Arthrobacter Ureafaciens'' (EC 3.2.1.18).
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<p>
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Characterisation information for the sialidase enzyme holds good for the following parts that constitute the sialidase enzyme sequence:
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<br>Basic part BBa_K2235005 consists of the sialidase enzyme coding sequence with a His tag C-terminally attached.
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<br>BBa_K2235006 biobrick has the RBS functional unit attached to sialidase part (BBa_K2235005) that can be used to test on various promoters.
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<br>BBa_K2235011 is a conjugation of sialidase composite (BBa_K2235009) with a device that facilitates the secretion of the enzyme out of the E.coli bacterial cell.
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<br>BBa_K2235007 biobrick constitutes OmpR fused to sialidase enzyme with RBS (BBa_K2235006).
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</p>
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</div>
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</html>
  
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This biobrick is a constitute of T7 promoter and RBS followed by sialidase enzyme coding site. Sialidase enzyme has the potential to digest terminal sialic acids in a glycoprotein. The sequence originates from the species ''Arthrobacter Ureafaciens''.
 
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==Usage and Biology==
 
==Usage and Biology==
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The sialidase enzyme was expressed from ''Arthrobacter Ureafaciens''. This enzyme hydrolyzes glycosidic linkages of terminal sialic acid residues in glycoproteins.
 
  
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Sialidase enzyme can hydrolyze glycosidic linkages of terminal sialic acid residues in glycoproteins. Figure-1 represents the reaction mechanism of an active enzyme.
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[[File:SiaComp_Mechanism.png|400px|thumb|left|Figure 1: Schematic representation of Sialidase enzyme reaction mechanism.]]
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==Characterization==
 
==Characterization==
  
 
===Important parameter===
 
===Important parameter===
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'''Table 1:''' Parameters used for expression and purification of sialidase enzyme.
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[[File:SiaComp_Parameter.png|400px|thumb|left|]]
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===Purification and identification===
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The cloned gblock plasmid culture was induced with IPTG for expression. Post expression the cells were sonicated and purified using Immobilized Metal Affinity Chromatography(IMAC). The purified enzymes were tested on SDS PAGE(Figure 2).
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As control, sialidase enzyme plasmid from literature research lab(see reference) was used. The control plasmid(au54) holds the enzyme coding site on a backbone which is not compatible with iGEM standards. Similar cloning, expression and purification methods were carried out on both, gblock+pSB1C3 plasmid and the control plasmid. A band could be observed at 60 kDa.
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Note: According to the literature the expected size of the enzyme should be at 55 kDa. However, the size of enzyme purified from the control plasmid(au54) and designed plasmid are consistent.
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[[File:SiaComp_SDSPAGE1.png|400px|thumb|left|Figure 2: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, the remaining three are IMAC purification fractions of sialidase.]]
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To demonstrate the above, an SDS-PAGE on both samples was carried out. The au54 sialidase and our designed biobrick sialidase, with molecular sizes of 54kDa and 55 kDa respectively. Figure 3 shows that there is no observable difference in size between the two proteins. Sialidase is a protein with a high content of basic amino acids, therefore it was hypothesized that this might affect the travelling speed through the gel.
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[[File:SiaComp_ladder.png|400px|thumb|left|Figure 3: SDS-PAGE gel and a protein ladder.From left to right: first well is a protein ladder, second well is protein expressed from the au54 plasmid, third well contains sialidase expressed from the biobrick plasmid.]]
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</p>
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===Hypothesis testing: Successfully expressed sialidase shows enzymatic activity on mucin ===
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<div style="text-align:justify">
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With the goal of testing the enzymatic activity of sialidase on mucin, an assay was developed and optimized. The objective was to measure the concentration of sialic acid released after digestion, which was quantified using high performance anion exchange chromatography (HPAEC).
 +
 +
Using industrially purchased sialidase to treat bovine submaxillary mucin (BSM) gave a positive result. Sialic acid was proved to be digested from the mucin. Therefore, the next step was to repeat the experiment with sialidase that we expressed in E. coli. A range of different sialidase concentrations were used and their respective sialic acid digestion quantified (figure 4). We were expecting a linear increase of substrate degraded with increase of enzyme. It is believed that excessive enzyme was used in the experiment. The sialic acid concentration released even at the lowest enzyme concentration is large when compared to the positive control (deglycosylation using sulfuric acid).
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</div>
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[[File:SiaComp_Hypothesis.png|600px|thumb|left|Figure 4: Concentrations of sialic acid digested from BSM in comparison to the concentration of sialidase used.]]
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===Rheology testing: Deglycosylated mucin samples show a decrease in viscosity===
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Removing glycans from mucins could induce protein backbone aggregation and thus decrease the viscosity. Due to time restraints, we chose to chemically deglycosylate PGM sample to simulate the enzymatic activity of sialidase for mucus degradation.Using a non-reducing chemical reagent mixture, we efficiently cleaved the glycans with minimal protein or glycan destruction.
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With rheology, we can measure the viscoelastic properties of a given structure. Our measurements showed that viscosity of deglycosylated compared to untreated PGM decreased at a higher rate with increasing shear rate (figure 5). Furthermore, this finding is supported by visually checking the fluidity of the PGM samples (figure 6).
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[[File:SiaComp_Rheo.png|600px|thumb|left|Figure 5: Rheology measurements of the changes in viscosity and stress of untreated mucin (native PGM) versus treated mucin (deglycosylated PGM).]]
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==Methods==
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===Cultivation===
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Ligation was done with ThermoFisher T4 DNA Ligase Buffer (10X) at 22 °C and the vectors were transformed into E. coli TOP10 and BL21(DE3) by heat shock. After growth overnight at 37 °C on petri dishes the results were documented. By analysing the number of colony forming units and color of the colonies the ligation efficiency of LigA should be assessable. For the negative control, there were no clones able to grow on chloramphenicol supplemented LB medium. For the positive control, a much bigger number of clones was observed when ligation was performed.
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[[File:SiaComp_culture.png|600px|thumb|left|Figure 6: Successful cultivation for Top10 and BL21 cells with BBa_K2235009.]]
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===Ligation of sialidase insert into pSB1C3 backbone===
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We cloned our gblock containing T7 promoter-RBS-sialidase with the compatible plasmid backbone (pSB1C3). Cloning of sialidase gBlock into a pSB1C3 vector was performed with T4 Ligase. To confirm successful cloning, we performed a double digest on the ligated plasmid. On gel electrophoresis we observed a band at ~1600 bp and one at ~2000 bp. The band at 1600 bp corresponds to the size of sialidase and the band at 2000 bp to the linearised plasmid backbone.
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[[File:SiaComp_3.png|600px|thumb|left|Figure 7:From left to right: M DNA ladder, next 2 lanes are plasmids digested with Ecor1 and Pst1.]]
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===IMAC purification===
  
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To purify the sialidase sample A1-A3, B1-B2 and control using the His-tag. IMAC purification was carried out using nickel column of volume 3.2mL and three cobalt columns, each with a volume of 1.2 mL. The protein samples was eluted to five fractions each.
  
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===Molecular cloning===
 
  
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'''Ligation of sialidase composite insert into pSB1C3'''
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The gblock containing T7 promoter-RBS-sialidase was ligated into the Chloramphenicol plasmid backbone (pSB1C3). Preliminary confirmation of cloning was done by double digest Ecor1 and Pst1. Figure 1 represents the double digestion result: bands were visible at ~1600 bp and ~2000 bp which represents the insert and plasmid backbone respectively.
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[[File:SiaComp_1.png|600px|thumb|left|Figure 1:
 
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From left to right: M DNA ladder, next 2 lanes are plasmids digested with Ecor1 and Pst1.]]
 
  
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==References==
  
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Egebjerg, J. and Christensen, S. (2005). Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens. Biotechnology and Applied Biochemistry, 41(3), p.225.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 19:55, 15 December 2017


Sialidase composite with T7 promoter and RBS

Introduction

This biobrick is a constitute of T7 promoter and RBS followed by the sialidase enzyme coding site. Sialidase enzyme has the potential to digest terminal sialic acids in a glycoprotein. The sequence originates from the species ''Arthrobacter Ureafaciens'' (EC 3.2.1.18).

Characterisation information for the sialidase enzyme holds good for the following parts that constitute the sialidase enzyme sequence:
Basic part BBa_K2235005 consists of the sialidase enzyme coding sequence with a His tag C-terminally attached.
BBa_K2235006 biobrick has the RBS functional unit attached to sialidase part (BBa_K2235005) that can be used to test on various promoters.
BBa_K2235011 is a conjugation of sialidase composite (BBa_K2235009) with a device that facilitates the secretion of the enzyme out of the E.coli bacterial cell.
BBa_K2235007 biobrick constitutes OmpR fused to sialidase enzyme with RBS (BBa_K2235006).

Usage and Biology

Sialidase enzyme can hydrolyze glycosidic linkages of terminal sialic acid residues in glycoproteins. Figure-1 represents the reaction mechanism of an active enzyme.

Figure 1: Schematic representation of Sialidase enzyme reaction mechanism.



Characterization

Important parameter

Table 1: Parameters used for expression and purification of sialidase enzyme.

SiaComp Parameter.png






Purification and identification

The cloned gblock plasmid culture was induced with IPTG for expression. Post expression the cells were sonicated and purified using Immobilized Metal Affinity Chromatography(IMAC). The purified enzymes were tested on SDS PAGE(Figure 2). As control, sialidase enzyme plasmid from literature research lab(see reference) was used. The control plasmid(au54) holds the enzyme coding site on a backbone which is not compatible with iGEM standards. Similar cloning, expression and purification methods were carried out on both, gblock+pSB1C3 plasmid and the control plasmid. A band could be observed at 60 kDa. Note: According to the literature the expected size of the enzyme should be at 55 kDa. However, the size of enzyme purified from the control plasmid(au54) and designed plasmid are consistent.

Figure 2: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, the remaining three are IMAC purification fractions of sialidase.

















To demonstrate the above, an SDS-PAGE on both samples was carried out. The au54 sialidase and our designed biobrick sialidase, with molecular sizes of 54kDa and 55 kDa respectively. Figure 3 shows that there is no observable difference in size between the two proteins. Sialidase is a protein with a high content of basic amino acids, therefore it was hypothesized that this might affect the travelling speed through the gel.

Figure 3: SDS-PAGE gel and a protein ladder.From left to right: first well is a protein ladder, second well is protein expressed from the au54 plasmid, third well contains sialidase expressed from the biobrick plasmid.




Hypothesis testing: Successfully expressed sialidase shows enzymatic activity on mucin

With the goal of testing the enzymatic activity of sialidase on mucin, an assay was developed and optimized. The objective was to measure the concentration of sialic acid released after digestion, which was quantified using high performance anion exchange chromatography (HPAEC). Using industrially purchased sialidase to treat bovine submaxillary mucin (BSM) gave a positive result. Sialic acid was proved to be digested from the mucin. Therefore, the next step was to repeat the experiment with sialidase that we expressed in E. coli. A range of different sialidase concentrations were used and their respective sialic acid digestion quantified (figure 4). We were expecting a linear increase of substrate degraded with increase of enzyme. It is believed that excessive enzyme was used in the experiment. The sialic acid concentration released even at the lowest enzyme concentration is large when compared to the positive control (deglycosylation using sulfuric acid).

Figure 4: Concentrations of sialic acid digested from BSM in comparison to the concentration of sialidase used.



Rheology testing: Deglycosylated mucin samples show a decrease in viscosity

Removing glycans from mucins could induce protein backbone aggregation and thus decrease the viscosity. Due to time restraints, we chose to chemically deglycosylate PGM sample to simulate the enzymatic activity of sialidase for mucus degradation.Using a non-reducing chemical reagent mixture, we efficiently cleaved the glycans with minimal protein or glycan destruction. With rheology, we can measure the viscoelastic properties of a given structure. Our measurements showed that viscosity of deglycosylated compared to untreated PGM decreased at a higher rate with increasing shear rate (figure 5). Furthermore, this finding is supported by visually checking the fluidity of the PGM samples (figure 6).

Figure 5: Rheology measurements of the changes in viscosity and stress of untreated mucin (native PGM) versus treated mucin (deglycosylated PGM).





Methods

Cultivation

Ligation was done with ThermoFisher T4 DNA Ligase Buffer (10X) at 22 °C and the vectors were transformed into E. coli TOP10 and BL21(DE3) by heat shock. After growth overnight at 37 °C on petri dishes the results were documented. By analysing the number of colony forming units and color of the colonies the ligation efficiency of LigA should be assessable. For the negative control, there were no clones able to grow on chloramphenicol supplemented LB medium. For the positive control, a much bigger number of clones was observed when ligation was performed.

Figure 6: Successful cultivation for Top10 and BL21 cells with BBa_K2235009.

Ligation of sialidase insert into pSB1C3 backbone

We cloned our gblock containing T7 promoter-RBS-sialidase with the compatible plasmid backbone (pSB1C3). Cloning of sialidase gBlock into a pSB1C3 vector was performed with T4 Ligase. To confirm successful cloning, we performed a double digest on the ligated plasmid. On gel electrophoresis we observed a band at ~1600 bp and one at ~2000 bp. The band at 1600 bp corresponds to the size of sialidase and the band at 2000 bp to the linearised plasmid backbone.

Figure 7:From left to right: M DNA ladder, next 2 lanes are plasmids digested with Ecor1 and Pst1.



IMAC purification

To purify the sialidase sample A1-A3, B1-B2 and control using the His-tag. IMAC purification was carried out using nickel column of volume 3.2mL and three cobalt columns, each with a volume of 1.2 mL. The protein samples was eluted to five fractions each.




References

Egebjerg, J. and Christensen, S. (2005). Cloning, expression and characterization of a sialidase gene from Arthrobacter ureafaciens. Biotechnology and Applied Biochemistry, 41(3), p.225.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 127
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 574
    Illegal NgoMIV site found at 649
    Illegal NgoMIV site found at 739
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1119