Part:BBa_K3037003
Fusion protein dCas9 + HRP (MBP/dCas9/linker/HRP/Strep-tag)
| Fusion protein | |
|---|---|
| Function | Colour detection of specific DNA sequences |
| Use in | Escherichia coli |
| RFC standard | Freiburg RFC25 standard |
| Backbone | pOCC97 |
| Submitted by | Team: TU_Dresden 2019 |
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 (recommended by Aliona ).. 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.
Overview of the full construct
Biology
In order to find more information regarding the Biology and function of our final construct, please check the registries of the single parts:
Characterization
Outline
We performed the following characterization experiments:
1) Expression in pOCC97 (BBa_K3037000):
The full construct, once all the single parts were fused together, was cloned into our expression plasmid K3037000 (p0CC97), and was expressed overnight. 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 and got two positive clones. On the right, the simulation of the digest in SnapGene is shown.
- T--TU Dresden--notebook-92.png
Overview of the full construct
https://2019.igem.org/wiki/images/1/13/
2) SDS-PAGEs for the expression assay over time (BBa_K3037003)
3) Image analysis of the expression in the SDS-PAGEs with imageJ
Experiments in Detail
1) Expression in pOCC97 (BBa_K3037000):
The fusion protein was expressed using the plasmid pOOC97 as a backbone (BB_aK3037000)
The purpose of this experiment is to show that the Escherichia coli grows normaly after the induction of the expression of the fusion protein.
For this the development of the culture was monitored by measuring the OD at 600 nm during different time 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.
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
SDS-PAGE (Check that the expression of the protein doesn't affect the growing of the bacterian)
Growing curve (Check that the expression of the protein doesn't affect the growing of the bacteria)
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)]
SDS-PAGE (Comparison of expression of different proteins in the vector before and after optimization)
Expression of full construct in pOCC97 not optimized at 18ÂșC
SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ÂșC before optimization)
Expression of Full Construct in pOCC97 not optimized at 37ÂșC
SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ÂșC before optimization)
SDS-PAGE (Expression of fusion protein BBa_K3037003 at 37ÂșC before optimization)
Expression of Full Construct in pOCC97 optimized
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 18ÂșC and at 37ÂșC
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
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
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Different temperature optimized 1mM
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Different induction concentration optimizedat 18 degrees
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Comparison optimized vs not optimized
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
Image analysis of SDS-PAGE (Expression of fusion protein BBa_K3037003)
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 18ÂșC and 0.5 mM IPTG. Also the optimization process is better than the not optimazed as the expression is more stable over time
Sequence
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2302
Illegal NheI site found at 5500 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 381
Illegal BglII site found at 5742
Illegal BamHI site found at 4581
Illegal BamHI site found at 5314
Illegal XhoI site found at 5826 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 79
References
| None |