Difference between revisions of "Part:BBa K3037003"

Revision as of 18:16, 20 October 2019

Fusion protein dCas9 + HRP (MBP/dCas9/linker/HRP/Strep-tag)

Overview

The TU Dresden 2019 team has designed this BioBrick in order to allow for the quick detection of specific DNA sequences of interest (more information).

The full construct is shown below, having each single marked part a specific function to optimize the full construct. The MBP and strep-tag allow purification via Amylose resin and via strep-columns, respectively. Additionally, the MBP enhances the expression of dCas9-fusion proteins and the linker helps in the folding process. The dCas9 identifies the sequence of interest and the HRP provides with a easy-detectable color-readout. More information regarding the biology, design and function of each basic part can be found here:

The single constructs were fused in PSB1C3 and PSB1A3 and finally the full construct was inserted into BBa_K3037000 vector for expression and characterization in Escherichia coli.

The weight of the protein was calculated based on the base pairs. 924 bp/3 = 308 amino acids, each amino acid weights as average 110 Dalton [1], so the final weight of approximately our construct is 230 kDa.

Growth curve of Expression in pOCC97, BBa_K3037000

Biology

In order to find more information regarding the Biology and function of our final construct, please check the registries of the single parts:

MBP: BBa_K3037001

dCas9: BBa_K3037002

Linker: BBa_K3037004

HRP: BBa_K3037007

Strep-tag: BBa_K823038

Characterization

Outline

We performed the following characterization experiments:

1) Expression of our Full Construct (FC) in pOCC97 (BBa_K3037000): and testing the growth of E.coli.

2) SDS-PAGEs showing the expression of the full construct over time

3) Analysis of the expression with ImageJ

4) Characterization of the single parts of the full construct

Experiments in Detail

1) Expression of our Full Construct (FC) in pOCC97 (BBa_K3037000): and testing the growth of E.coli.

The full construct, once all the single parts were fused together, was cloned into our expression plasmid K3037000 (p0CC97). The correct insertion of our full construct into the plasmid was proven via restriction digest followed by agarose gel electrophoresis. For that, we performed a triple digest with PmlI, X and P and got several positive clones. On the right, the simulation of the digest in SnapGene is shown.

Multiple full construct clones digested with PmlI, XbaI and Pst-1. On the right, the simulation in SnapGene is shown. The positive clones are marked with red crosses.

Furthermore, it was proven that the E. coli could grow normally after the induction of the fusion protein. For that, the development of the bacteria cultures was monitored by measuring the OD at 600 nm during different time points before and after induction with 1 mM IPTG.

As shown in the curve, the growth of the bacteria is not affected by the expression of the protein.

Comparison of the growth curve compared before and after optimization

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  Growing curve (Check that the expression of the protein doesn't affect the growing of the bacteria)

Expression in BBa_K3037000 at different temperatures and comparison between the pOOC97 optimized and not optimized

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  SDS-PAGE (Check that the expression of the protein doesn't affect the growing of the bacterian)

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  Growing curve (Check that the expression of the protein doesn't affect the growing of the bacteria)

Comparison of the growth curve compared before and after optimization

2) SDS-PAGEs showing the expression assay over time

After proving that the final construct was well inserted in our plasmid, the full construct was expressed overnight. The first expression was performed at 37°C for seven hours, induced with 1 mM IPTG. The result is shown below:

SDS-page showing the expression of the full construct in POCC97 after induction with 1 mM IPTG. Different type points show the increased expression of the full construct (marked with black arrow).

3) Image analysis of the expression in the SDS-PAGEs with imageJ

2) SDS-PAGEs for the expression assay over the time of Full Construct (BBa_K3037003)

Comparison of the expression of MBP-HRP (BBa_K3037008) and Full Construct https://parts.igem.org/Part:BBa_K3037003 (BBa_K3037003)]

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  SDS-PAGE (Comparison of expression of different proteins in the vector before and after optimization)

Expression of full construct in pOCC97 not optimized at 18ºC

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  SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ºC before optimization)

Expression of Full Construct in pOCC97 not optimized at 37ºC

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  SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ºC before optimization)

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  SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ºC before optimization)

Expression of Full Construct in pOCC97 optimized

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  SDS-PAGE (Expression of fusion protein BBa_K3037003 after optimization)

3) Image analysis of the expression in the SDS-PAGEs with imageJ

Different induction concentration not optimized at 18ºC and at 37ºC

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Not optimized at different temperature at 0.2 mM, 0.5 mM and 1 mM IPTG expression induction

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Different temperature optimized 1mM

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Different induction concentration optimizedat 18 degrees

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Comparison optimized vs not optimized

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

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  Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)

Conclusion

Based on this analysis can be concluded that optimal conditions for the expression of BBa_3037003 are 18ºC and 0.5 mM IPTG. Also the optimization process is better than the not optimazed as the expression is more stable over time

4) Strep-tag column purification

This BioBrick include the Strep-tag BioBrick BBa_K823038.

This was used for make a purification of the fusion protein using the “Expression and purification of proteins using Strep-Tactin” protocol of IBA Lifescience. The results of the following gel shows that the purification doesn't worked. We thought that this is because the Strep-tag that we used is meant to be used for Western Blots and not for column purification.

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  Strep-tactin column purification (Purification of the composite BioBrick BBa_K3037003)

Sequence

References

[1] https://www.promega.com/~/media/files/resources/technical%20references/amino%20acid%20abbreviations%20and%20molecular%20weights.pdf