Difference between revisions of "Part:BBa K2235010"

(→‎Molecular Cloning)
(→‎Reference)
 
(19 intermediate revisions by 2 users not shown)
Line 9: Line 9:
  
 
==Usage and Biology==
 
==Usage and Biology==
−
The endo-β-galactosidase (Endo-β-GalGnGa) was originally expressed from Clostridium perfringens. This bacteria strain is capable of releasing GlcNAcα1→4Gal from glycans expressed in the gastric mucous cell-type mucin [1].
 
−
This enzyme specifically releases the disaccharide GlcNAcR1f 4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin. This enzyme has been shown to hydrolyze the endo-â-galactosyl linkage not only in the GlcNAcR1f 4Galâ1f4GlcNAc sequence but also in GlcNAcR1f 4Galâ1f3GalNAcR1fSer/Thr. Endo-â-GalGnGa is distinct from the hitherto known endo-â-galactosidases because of its strict specificity for releasing the disaccharide GlcNAcR1f 4Gal. To characterize Endo-â-GalGnGa, we have carried out the molecular cloning of this endoglycosidase. Here we describe the cloning, characterization, and overexpression of the gene encoding Endo-â-GalGnGa and the hypothesis testing of degrading mucus.
 
  
−
==Important parameters==
+
[[File:EBGComp_Mechanism.png|600px|thumb|left|Figure 1: Schematic representation of EBG enzyme reaction mechanism.]]
  
−
==Results==
+
The endo-β-galactosidase (EBG) was originally expressed from Clostridium perfringens. This bacteria strain is capable of releasing GlcNAcα1→4Gal from glycans expressed in the gastric mucous cell-type mucin [1].This enzyme specifically releases the disaccharide GlcNAcR1f 4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin. EBG is distinct from the hitherto known endo-â-galactosidases because of its strict specificity for releasing the disaccharide GlcNAcR1f 4Gal. To characterize EBG, we have carried out the molecular cloning of this endoglycosidase. Here we describe the cloning, characterization, and overexpression of the gene encoding EBG and the hypothesis testing of degrading mucus.
−
After confirming that the cloning worked, the plasmid was transformed into E. coli BL21(DE3) and expression was induced at multiple combinations of OD600 and IPTG concentrations. The expression of EBG, a 47 kDa protein, from one of the successful expressions (at OD600 of 0.4 and an IPTG concentration of 0.5 mM) is shown in figure 2.
+
  
−
Figure 2 is attached here
 
  
  
−
By carrying out the experiments above we demonstrated successful cloning and expression of both of our mucus degrading enzymes: sialidase and EBG. The next step was to secrete the enzymes, using an already existing biobrick for HylA E.coli secretion system (BBa_K1166002) from the iGEM 2017 distribution kit. Firstly, we removed the stop codon at the end of the sialidase gblock sequence using PCR and thereafter cloned sialidase without the stop codon upstream of the secretion system. The newly cloned plasmid was transformed into E.coli and expression in flask was induced with 0.5 mM IPTG. The enzyme was extracted from the medium using IMAC purification. Figure 3 shows no secretion of a protein resembling the correct size (≈ 55 kDa).
+
==Characterizations==
  
−
Figure 3 is attached here
+
===Important Parameters===
  
−
==Molecular Cloning==
+
[[File:EBGComp_Parameter.png|600px|thumb|left|Table 1: Parameters used for expression and purification of EBG enzyme.]]
−
'''Ligation of EBG insert into iGEM backbone''' 
+
  
−
We designed our endo-β-galactosidase (EBG) biobrick (BB_XXX) by modifying the sequence of professor Li (2002,). The sequence received did not contain a His6-tag, which was added to the sequence for later IMAC purification steps. The biobrick was cloned into an iGEM compatible plasmid backbone (pSB1C3). To confirm successful cloning, we double digested plasmids from five different colonies and the results are shown in figure 4. For each colony, two bands could be observed. One at ~1400 bp, corresponding to the size of EBG, and one at ~2000 bp, corresponding to the size of the plasmid backbone.
+
<p align="justify">
  
−
Figure 4 is attached here
+
</p>
  
  
−
'''Cultivation'''
+
<br style="clear: both" />
  
−
Cloning of EBG gBlock into a pSB1C3 vector was performed with T4 Ligase. 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.
+
===Purification and Identification of EBG===
  
−
Figure 5 is attached here
+
After confirming that the cloning worked, the biobrick plasmid (BBa_K2235010) was transformed into E. coli BL21(DE3) and expression was induced at multiple combinations of OD600 and IPTG concentrations. SDS-PAGE results from one of the successful expressions (at OD600 of 0.4 and an IPTG concentration of 0.5 mM) show the expression of EBG, a 47 kDa protein (figure 2). The band on lane three is believed to be EBG.
  
−
By analyzing 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.
+
[[File:EBGComp_2.png|600px|thumb|left|Figure 2: SDS-PAGE gel and a protein ladder. From left to right: protein ladder, the remaining five are IMAC purification fractions of EBG.]]
 +
 
 +
<p align="justify">
 +
 
 +
</p>
 +
 
 +
 
 +
<br style="clear: both" />
 +
 
 +
===EBG Enzymatic Activity on Mucin===
 +
To determine the concentration of sugars after the EBG digestion of PGM a colorimetric assay was performed. Industrially available EBG was used firstly to test the assay. The results from the assay were inconclusive as a result of a strong response from the negative control.
 +
 
 +
Due to time restraints this part of the project was never completed. The next step after fixing the assay would have been to test the expressed EBG from ''E. coli''.
  
 
==Methods==
 
==Methods==
 +
===IMAC purification===
  
−
IMAC purification
+
To purify the EBG samples and the control based on the containing Histag. The protocol was used with no changes. The colons used for the IMAC purification was one nickel colon with colon volume of 3,2 mL and three cobalt colons, each with colon volumes of 1,2 mL. The protein samples was eluted to five fractions each. SDS-PAGE was performed after IMAC purification to visualize the expressed EBG samples
−
Aim
+
−
To purify the EBG samples and the control based on the containing Histag.
+
  
−
Procedure
 
−
The protocol was used with no changes. The colons used for the IMAC purification was one nickel colon with colon volume of 3,2 mL and three cobalt colons, each with colon volumes of 1,2 mL. The protein samples was eluted to five fractions each.
 
  
−
SDS-PAGE and staining
+
===Ligation of sialidase insert into iGEM backbone===
−
Aim
+
 
−
To visualize the expressed Sialidase samples and make sure the correct protein has been expressed by comparing the size to the ladder on the SDS-PAGE gel.
+
We designed our endo-β-galactosidase (EBG) biobrick (BBa_K2235010) by modifying the sequence of professor Li (Ashida, 2002). The sequence received did not contain a His6-tag, which was added to the sequence for later IMAC purification steps. The biobrick containing T7 promoter-RBS-EBG was cloned into an iGEM compatible plasmid backbone (pSB1C3). To confirm successful cloning, we double digested plasmids from five different colonies (figure 3). For each colony, two bands could be observed. One at ~1400 bp, corresponding to the size of EBG, and one at ~2000 bp, corresponding to the size of the plasmid backbone.
 +
 
 +
[[File:EBGCompo_1.png|600px|thumb|left|Figure 3: Agarose gel. From left to right: DNA ladder, empty well, the next five are digested BBa_K2235010 plasmids.]]
 +
 
 +
<p align="justify">
 +
 
 +
</p>
 +
 
 +
 
 +
<br style="clear: both" />
 +
 
 +
===Cultivation===
 +
Cloning of EBG gBlock into a pSB1C3 vector was performed with T4 Ligase. 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 analyzing the number of colony forming units and color of the colonies the ligation efficiency of LigA should be accessible. 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.
  
−
Procedure
+
[[File:EBGCompo_Culture.png|600px|thumb|left|Figure 4: Successful cultivation for Top10 cells with BBa_K2235010.]]
−
The protocol for SDS-PAGE used with some modifications.
+
  
−
Protein samples and loading buffer were mixed at a ratio of 24 µL of protein sample and 6 µL of loading buffer in a fume hood. Loading buffer was prepared by staff at floor 2.
+
<p align="justify">
−
A pre-casted gel was used (Mini-Protean TGX from BIORAD).
+
  
−
The samples that were run on SDS-PAGE were the IMAC purified A1-A3, B1-B2 and C1-C3. B3 and the control were not run because lack of space of the gels. These samples were run later on SDS-PAGE gel. The gels were stained using the protocol for SimplyBlue SafeStain.
+
</p>
  
  
 +
<br style="clear: both" />
  
 
==Reference==
 
==Reference==
−
1. Ashida, H., Anderson, K., Nakayama, J., Maskos, K., Chou, C.-W., Cole, R. B., Li, S.-C., and Li, Y.-T. (2001) J. Biol. Chem. 276, 28226−28232
+
1. Ashida, H., Maskos, K., Li, S. and Li, Y. (2002). Characterization of a Novel Endo-β-galactosidase Specific for Releasing the Disaccharide GlcNAcα1→4Gal from Glycoconjugates†,‡. Biochemistry, 41(7), pp.2388-2395.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 21:05, 31 October 2017


Endo beta galactosidase with T7 promoter, RBS and functional unit

Introduction

BBa_K2235010 biobrick is a composite of a T7 promoter and RBS followed by endo-β-galactosidase enzyme coding site which is N-terminally attached to a His-tag. This enzyme has been shown to release saccharide chains from glycans expressed in gastric mucus. It performs this action by hydrolysing the bonds next to galactose saccharides in the polysaccharide chains of mucins. The sequence originates from the species Clostridium Perfringens. The basic part BBa_K2235008 consists of the endo beta galactosidase enzyme coding sequence with a His-tag N-terminally attached.


Usage and Biology

Figure 1: Schematic representation of EBG enzyme reaction mechanism.

The endo-β-galactosidase (EBG) was originally expressed from Clostridium perfringens. This bacteria strain is capable of releasing GlcNAcα1→4Gal from glycans expressed in the gastric mucous cell-type mucin [1].This enzyme specifically releases the disaccharide GlcNAcR1f 4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin. EBG is distinct from the hitherto known endo-â-galactosidases because of its strict specificity for releasing the disaccharide GlcNAcR1f 4Gal. To characterize EBG, we have carried out the molecular cloning of this endoglycosidase. Here we describe the cloning, characterization, and overexpression of the gene encoding EBG and the hypothesis testing of degrading mucus.


Characterizations

Important Parameters

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



Purification and Identification of EBG

After confirming that the cloning worked, the biobrick plasmid (BBa_K2235010) was transformed into E. coli BL21(DE3) and expression was induced at multiple combinations of OD600 and IPTG concentrations. SDS-PAGE results from one of the successful expressions (at OD600 of 0.4 and an IPTG concentration of 0.5 mM) show the expression of EBG, a 47 kDa protein (figure 2). The band on lane three is believed to be EBG.

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



EBG Enzymatic Activity on Mucin

To determine the concentration of sugars after the EBG digestion of PGM a colorimetric assay was performed. Industrially available EBG was used firstly to test the assay. The results from the assay were inconclusive as a result of a strong response from the negative control.

Due to time restraints this part of the project was never completed. The next step after fixing the assay would have been to test the expressed EBG from E. coli.

Methods

IMAC purification

To purify the EBG samples and the control based on the containing Histag. The protocol was used with no changes. The colons used for the IMAC purification was one nickel colon with colon volume of 3,2 mL and three cobalt colons, each with colon volumes of 1,2 mL. The protein samples was eluted to five fractions each. SDS-PAGE was performed after IMAC purification to visualize the expressed EBG samples


Ligation of sialidase insert into iGEM backbone

We designed our endo-β-galactosidase (EBG) biobrick (BBa_K2235010) by modifying the sequence of professor Li (Ashida, 2002). The sequence received did not contain a His6-tag, which was added to the sequence for later IMAC purification steps. The biobrick containing T7 promoter-RBS-EBG was cloned into an iGEM compatible plasmid backbone (pSB1C3). To confirm successful cloning, we double digested plasmids from five different colonies (figure 3). For each colony, two bands could be observed. One at ~1400 bp, corresponding to the size of EBG, and one at ~2000 bp, corresponding to the size of the plasmid backbone.

Figure 3: Agarose gel. From left to right: DNA ladder, empty well, the next five are digested BBa_K2235010 plasmids.



Cultivation

Cloning of EBG gBlock into a pSB1C3 vector was performed with T4 Ligase. 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 analyzing the number of colony forming units and color of the colonies the ligation efficiency of LigA should be accessible. 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 4: Successful cultivation for Top10 cells with BBa_K2235010.



Reference

1. Ashida, H., Maskos, K., Li, S. and Li, Y. (2002). Characterization of a Novel Endo-β-galactosidase Specific for Releasing the Disaccharide GlcNAcα1→4Gal from Glycoconjugates†,‡. Biochemistry, 41(7), pp.2388-2395.

Sequence and Features



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 246
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]