Difference between revisions of "Part:BBa K3794005"
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<h2> DNA Amplification and Purification </h2>
+
<h2> DNA Amplification and DNA Purification </h2>
  
 
Due to the large size of this composite part, 3322bp, it was synthesised in two fragments by IDT.  
 
Due to the large size of this composite part, 3322bp, it was synthesised in two fragments by IDT.  
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Following PCR, ChABC DNA from the Agarose gel was excised and purified and stored at -20C.
 
Following PCR, ChABC DNA from the Agarose gel was excised and purified and stored at -20C.
  
−
<h2> Protein expression </h2>
+
<h2> Protein Expression </h2>
  
 
ChABC was ligated into pSB1A3 using XbaI and SpeI, and this expression vector was transformed into competent E.coli BL21 (DE3) cells for protein expression through IPTG-induction at 37C.
 
ChABC was ligated into pSB1A3 using XbaI and SpeI, and this expression vector was transformed into competent E.coli BL21 (DE3) cells for protein expression through IPTG-induction at 37C.
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<h2> Purification </h2>  
+
<h2> Protein Purification </h2>  
  
 
A Ni-NTA spin column (Qiagen) was used to purify our expressed ChABC protein (which corresponds to BBa_K3794004). Protein purification was conducted under native conditions
 
A Ni-NTA spin column (Qiagen) was used to purify our expressed ChABC protein (which corresponds to BBa_K3794004). Protein purification was conducted under native conditions
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The yield of our purified protein was very very low !!!!!!!!
+
As seen in Figure 5, no conclusive bands that can be inferred to be ChABC (~115kDa) can be seen in either sample - especially in the purified samples.
  
−
Isoelectric point !!!
+
Conducting literature reviews following this, we learnt that the isoelectric point of ChABC (pH 6.99) is very similar to the pH of our SDS-PAGE stacking gel (6.8). Heating our ChABC samples prior to loading onto SDS-PAGE gel potentially exposes hydrophobic regions which can cause aggregation on the gel.
 +
 
 +
Running a gel with non-heated samples improved our results (Figure 6). Here we can see purified ChABC, albeit in a low yield.
 +
 +
Figure 6
  
 
<h3> Western Blotting </h3>
 
<h3> Western Blotting </h3>
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Western Blot results indicated presence of 6xHis tagged ChABC as shown below.  
 
Western Blot results indicated presence of 6xHis tagged ChABC as shown below.  
  
−
Figure 6.
+
Figure 7.
  
  
−
<h2> Protein expression </h2>  
+
<h2> Protein Expression 2.0 </h2>  
  
 
Following a low yield of purified protein, and inconclusive expression results. We used competent BL21 E.coli instead of their DE3 counterparts in an effort to improve soluble protein yield. Following expression using BL21 and induction with IPTG, we ran an SDS-PAGE gel to see the results of our new expression system.  
 
Following a low yield of purified protein, and inconclusive expression results. We used competent BL21 E.coli instead of their DE3 counterparts in an effort to improve soluble protein yield. Following expression using BL21 and induction with IPTG, we ran an SDS-PAGE gel to see the results of our new expression system.  
Line 66: Line 70:
 
As it can be seen below, using BL21 E.coli slightly improved the yield of ChABC in the soluble fraction of the cell-lysate.  
 
As it can be seen below, using BL21 E.coli slightly improved the yield of ChABC in the soluble fraction of the cell-lysate.  
  
 +
Figure 8
  
−
<h2> Protein Purification </h2>  
+
<h2> Protein Purification 2.0 </h2>  
  
 
Having slightly improved the yield of our soluble protein, we conducted another protein purification, however instead using a Ni-NTA resin (ThermoFisher) rather than a Spin column (Qiagen).  
 
Having slightly improved the yield of our soluble protein, we conducted another protein purification, however instead using a Ni-NTA resin (ThermoFisher) rather than a Spin column (Qiagen).  
Line 73: Line 78:
 
The results of our second protein purification can be seen below  
 
The results of our second protein purification can be seen below  
  
−
Figure 7.  
+
Figure 9.  
  
−
Similar to previous results, the yield of our purified protein still remained low.
+
Similar to previous results, the yield of our purified protein still remained low. There are several reasons for this; due to the large size of ChABC (~115kDa), it may be that the Ni-NTA column is getting oversaturated with protein, and unable to accommodate a sufficient yield. Another possibility may be that the mutations we have introduced affect the solubility of ChABC, or that E.coli may not be sufficient hosts for the protein as ChABC is already a bacterial enzyme derived from <i> Proteus vulgaris </i>. 
  
  
 
<h2> Enzymatic Assay </h2>  
 
<h2> Enzymatic Assay </h2>  
  
−
Despite having a low yield of purified ChABC, we attempted to conduct an enzymatic assay to prove that our recombinant protein is functional.  
+
Despite having a low yield of purified ChABC, we attempted to conduct an enzymatic assay to prove that our recombinant protein is functional at 37C.
  
 
Following concentration of our eluted protein samples, and a buffer exchange using 15mM Tris-HCl we were able generate a concentrated protein sample of 0.014mg/mL
 
Following concentration of our eluted protein samples, and a buffer exchange using 15mM Tris-HCl we were able generate a concentrated protein sample of 0.014mg/mL

Revision as of 23:58, 19 October 2021


ChABC Composite

This composite part acts as a translational unit for the expression of ChABC in E.coli. The LacI regulatory system (BBa_R0010) which encodes the lac promoter and operator allow for IPTG-induced expression in E.coli. BBa_B0010, a T1 terminator from E.coli rrnB also allows for termination of transcription. As the ChABC basic part encodes a 6xHis tag and TEV cleavage site, protein purification using a Ni-NTA column can be achieved after expression of ChABC using this composite part.

This composite part can be utilised with any RFC[10] compatible plasmid backbone.

This part, BBa_K3794005, was directly synthesised by IDT.


Usage and Biology

Introduction


DNA Amplification and DNA Purification

Due to the large size of this composite part, 3322bp, it was synthesised in two fragments by IDT.

PCR using a thermal cycler was used for the amplification of both fragments of this composite part.

PCR products were confirmed using a 1% Agarose gel electrophoresis:

Figure 1 Figure 2


Following PCR, ChABC DNA from the Agarose gel was excised and purified and stored at -20C.

Protein Expression

ChABC was ligated into pSB1A3 using XbaI and SpeI, and this expression vector was transformed into competent E.coli BL21 (DE3) cells for protein expression through IPTG-induction at 37C.

A SDS-PAGE gel analysis of these expression results are shown below in Figure 3(lane 2-5).


There were no distinct, conclusive bands pointing towards expression of 6xHis tagged ChABC from the samples taking during protein expression. However, we proceeded to conduct a protein purification using a Qiagen Ni-NTA Spin column under native conditions


Protein Purification

A Ni-NTA spin column (Qiagen) was used to purify our expressed ChABC protein (which corresponds to BBa_K3794004). Protein purification was conducted under native conditions

An SDS-PAGE of purification can be seen below in Figure 5.

Figure 5


As seen in Figure 5, no conclusive bands that can be inferred to be ChABC (~115kDa) can be seen in either sample - especially in the purified samples.

Conducting literature reviews following this, we learnt that the isoelectric point of ChABC (pH 6.99) is very similar to the pH of our SDS-PAGE stacking gel (6.8). Heating our ChABC samples prior to loading onto SDS-PAGE gel potentially exposes hydrophobic regions which can cause aggregation on the gel.

Running a gel with non-heated samples improved our results (Figure 6). Here we can see purified ChABC, albeit in a low yield.

Figure 6

Western Blotting

Following protein purification, a western blot analysis was conducted to confirm the presence of 6xHis ChABC (BBa_K3794004) (which is a result of the expression from composite part BBa_K3794005).

Western Blot results indicated presence of 6xHis tagged ChABC as shown below.

Figure 7.


Protein Expression 2.0

Following a low yield of purified protein, and inconclusive expression results. We used competent BL21 E.coli instead of their DE3 counterparts in an effort to improve soluble protein yield. Following expression using BL21 and induction with IPTG, we ran an SDS-PAGE gel to see the results of our new expression system.

As it can be seen below, using BL21 E.coli slightly improved the yield of ChABC in the soluble fraction of the cell-lysate.

Figure 8

Protein Purification 2.0

Having slightly improved the yield of our soluble protein, we conducted another protein purification, however instead using a Ni-NTA resin (ThermoFisher) rather than a Spin column (Qiagen).

The results of our second protein purification can be seen below

Figure 9.

Similar to previous results, the yield of our purified protein still remained low. There are several reasons for this; due to the large size of ChABC (~115kDa), it may be that the Ni-NTA column is getting oversaturated with protein, and unable to accommodate a sufficient yield. Another possibility may be that the mutations we have introduced affect the solubility of ChABC, or that E.coli may not be sufficient hosts for the protein as ChABC is already a bacterial enzyme derived from Proteus vulgaris .


Enzymatic Assay

Despite having a low yield of purified ChABC, we attempted to conduct an enzymatic assay to prove that our recombinant protein is functional at 37C.

Following concentration of our eluted protein samples, and a buffer exchange using 15mM Tris-HCl we were able generate a concentrated protein sample of 0.014mg/mL




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2954
  • 1000
    COMPATIBLE WITH RFC[1000]