Difference between revisions of "Part:BBa K3037003"

Revision as of 11:44, 21 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.

Visualization of the full construct with its single parts.

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

However, the theoretical analysis of the expected parameters of the new protein (determined with ExPASy ProtParam tool) are the followings:

Extinction coefficient: 205750 L/(mol*cm)

Estimated half-life > 30 hours in mammalian cells, >20 hours in yeast, >10 hours in E. coli.

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 assay over time

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

4) Purification of the Full Construct with MBP-tag

5) 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. Important to note here is that the expression of the full construct was performed in two slightly different POCC97 plasmids, that differ on their Ribosome Binding Site (RBS). Hereinafter they are going to be referred to as optimized and not optimized (read the registry page BBa_K3037003 to for more details regarding the difference between these two plasmids(LINK!)).

Comparison of the growth curve compared before and after optimization

To go further, the expression of the full construct in pOCC97 at different temperatures was studied. For that, the optimized and not optimized pOOC97 were compared.

Comparison of the growth curves of optimized and not optimized pOCC97.

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).

The same experiment was repeated several times at different temperatures and IPTG concentrations in both, the optimized and not optimized POCC97 to compare the best expression conditions. The results are shown in the Figures below.

Expression of Full Construct in pOCC97 not optimized at 18ºC and different IPTG concentrations

Expression of the Full Construct in not optimized pOCC97 at 18ºC

Expression of Full Construct in pOCC97 not optimized at 37ºC and different IPTG concentrations

Expression of the Full Construct in not optimized pOCC97 at 37ºC

Expression of the Full Construct in pOCC97 optimized at different temperatures and IPTG concentrations

Expression of the Full Construct in optimized pOCC97 at different temperatures and IPTG concentrations

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

The previously shown SDS-pages were then further analysed by using the software ImageJ to correct for loading differences and be able to draw conclusions about the best conditions to express the Full Construct in POCC97.

Temperature and IPTG induction dependence of the optimized POCC97

Expression of the Full Construct in optimized pOCC97 under different conditions.

Temperature and IPTG induction dependence of the not optimized POCC97

Expression of the Full Construct in not optimized pOCC97 under different conditions.

Comparison between optimized and not optimized POCC97

Comparison between the expression of optimized and not optimized POCC97.

Conclusion

Based on this analysis it can be concluded that the optimal conditions for the expression of BBa_3037003 in POCC97 are 18ºC and 0.5 mM IPTG. The expression seems to be more stable over time for the optimized plasmid than for the non-optimized.

4) Purification of the Full Construct

After proving that the Full Construct is expressed properly in our plasmid and improved its expression conditions, it was purified by using Amylose Resin to bind its MBP site. To test for the correct functioning of each single part of the fusion protein we performed different experiments. For that, two different protocols were used. On the one hand, an Amylose Resin column was used and on the other hand a batch binding solution was prepared. Better results were obtained with the latter one and therefore, in which the resin was pipetted into a falcon and incubated with the cell lysate for 1.5 hours on a rotator at 4°C. The purification is shown in the following Figure.

5) Activity assay of the HRP

Investigating the activity of HRP in the full construct was done in a dynamic assay. The absorbance at 650 nm was measured over time with the substrate TMB (which is colorless and converted into a blue product by oxidation through HRP.

The HRP used for the comparison was the BBa_K1800002

Comparison of the activity

6) Strep-tag column purification

The reason to include a Strep-tag at the end of our Full Construct was to facilitate its purification. However, as already explained in the Registry page of the Strep-tag itself BBa_K823038, this BioBrick seems to not be working properly for column purification. The Strep-tag is probably meant to be used for Western Blots and not for column purification. That is why the purification via Strep-tag did not work (see Figure below). However, it was shown in the section before, that we were able to successfully purify it via the MBP.

Different Full Construct samples before, while and after Strep purification

7) Characterization of the single parts of the full construct

MBP

As already mentioned, the Full Construct was successfully purified by using Amylose Resin to bind its MBP site.

BBa_K3037001

dCas9

BBa_K3037002

Linker

BBa_K3037004

HRP

To test for the proper activity of the HRP in the Full Construct we performed enzymatic activity assays, as the ones performed to characterize the HRP BioBrick BBa_K3037007.

Sequence

NOTE: For some reason the specified scar when aploaded the sequence was the one of the RFC25 standard but the registry shows the one of the RFC23

References

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