Difference between revisions of "Part:BBa K3794005"
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<h2> Introduction </h2> | <h2> Introduction </h2> | ||
| + | <h2> Modelling </h2> | ||
<h2> DNA Amplification and DNA Purification </h2> | <h2> DNA Amplification and DNA Purification </h2> | ||
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PCR using a thermal cycler was used for the amplification of both samples of this composite part. | PCR using a thermal cycler was used for the amplification of both samples of this composite part. | ||
| − | PCR products were confirmed using a 1% Agarose gel electrophoresis | + | Correct PCR products were confirmed using a 1% Agarose gel electrophoresis as strong bands appear around the 3000bp region. |
<center> | <center> | ||
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</figure> | </figure> | ||
<figcaption> | <figcaption> | ||
| − | <b>Figure 1 | + | <b>Figure 1              Figure 2 </b> |
</figcaption> | </figcaption> | ||
</center> | </center> | ||
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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 | ||
| − | An SDS-PAGE of purification can be seen below in Figure | + | An SDS-PAGE of purification can be seen below in Figure 4. |
| − | Figure | + | Figure 4 |
| − | As seen in Figure | + | As seen in Figure 4, 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. | 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 | + | Running a gel with non-heated samples improved our results (Figure 5). Here we can see purified ChABC, albeit in a low yield. |
| − | Figure 6 | + | <center> |
| + | <figure> | ||
| + | <img width="300px" src="https://static.igem.org/mediawiki/parts/2/2f/T--KCL_UK--ChABCPur3.png"> | ||
| + | </figure> | ||
| + | <figcaption> | ||
| + | <b>Figure 5</b> SDS-PAGE gel resulting from protein purification of expressed ChABC. Lane 1: Promega Broad Range Protein Molecular Weight Markers. Lane 2: Cell-lysate pellet resuspended with 6M GuHCl. Lane 3: Elution 1. Lane 4: Elution 2. Lane 5: Elution 3. Lane 6: Elution 4. | ||
| + | </figcaption> | ||
| + | </center> | ||
| + | <br></br> | ||
| + | |||
<h3> Western Blotting </h3> | <h3> Western Blotting </h3> | ||
Revision as of 02:10, 20 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
Modelling
DNA Amplification and DNA Purification
Due to the large size of this composite part, 3322bp, it was synthesised in two samples. PCR using a thermal cycler was used for the amplification of both samples of this composite part. Correct PCR products were confirmed using a 1% Agarose gel electrophoresis as strong bands appear around the 3000bp region.
Figure 2: SnapGene simulation of 1% Agarose gel electrophoresis. Lane 1: Promega 1kb DNA Ladder. Lane 2: -- Lane 3: ChABC Sample 1. Lane 4: ChABC Sample 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. A band should appear around the 120kDa region to demonstrate presence of ChABC. Despite this, 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 4. Figure 4 As seen in Figure 4, 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 5). Here we can see purified ChABC, albeit in a low yield.