Difference between revisions of "Part:BBa K1800002"

Overview

This BioBrick was adapted to the Freiburg RFC25 standard by the Team: TU_Dresden 2019 and their characterization was implemented in this page. Also you can find it here: BBa_K3037007

Description

Class III plant peroxidases catalyze various oxidative reactions in which electrons are transferred to peroxide species, and substrate molecules are oxidized (Krainer, 2015). Peroxidases can be found in most plants and have been proposed to influence various functions related to the degradation of indole-3-acetic acid (IAA) (Lamport, 1986) and cell wall elasticity (Goldberg et al., 1986). Horseradish peroxidases is a peroxidase that has been used exhaustively as a reporter enzyme in diagnostics and histochemistry. In our lab, we designed an HRP part in accordance to RFC 10. HRP in our experiment will serve as a proof of concept for the Agrobacterium tumefaciens mediated transformation of tobacco plants with CBDA synthase. HRP will be inserted into the pORE vector for transformation of A. tumefaciens. The A. tumefaciens will then be used to transform tobacco plants. The tobacco will then produce hairy roots suspended in the culture that contains the HRP part.

Characterization: TU_Dresden 2019

Outline

We performed the following characterization experiments:

1) Growth curve of expression in pOCC97 (BBa_K3037000) in E. coli pRARE T7

2) Determination of the total protein concentration of cleared lysate after expression assay of the substrate conversion (TMB) compared to (BBa_K1800002)

3) Protein Expression monitored via SDS-PAGE

4) Activity assay of the substrate conversion TMB compared to (BBa_K1800002)

Experiments in Detail

1) Growth curve of expression in pOCC97 (BBa_K3037000) in E. coli pRARE T7

The HRP was expressed using the plasmid pOCC97 as a backbone (BBa_K3037000)

The purpose of this experiment was to show that the Escherichia coli pRARE T7 grows normally after the induction of HRP expression.

For this, growth of bacteria was monitored by measuring the Optical absorbance at 600 nm during different time points before and after induction with 1 mM IPTG. As shown in the following Figure, the Escherichia coli growth is not affected by the expression of the protein. It shows a normal growth behaviour as expected in a batch culture (Figure 1).

Figure 1: Growth curve of Escherichia coli before and after expressing HRP. Vector pOCC97 (BBa_K3037000). Induction with 1mM IPTG

2) Determination of the total protein concentration of cleared lysate after expression assay of the substrate conversion (TMB) compared to (BBa_K1800002)

In order to determine the total protein content of the cleared lysate after the expression the following assay was performed. First, the standard curve was done with the Pierce BCA protein assay kit of Thermo Scientific (#23225) (Figure 2).

Figure 2: Calibration curve with known BSA concentrations in order to determine the concentrations of our samples.

Then different cultures of E. coli pRARE T7 transformed with different BioBricks using pOCC97 (BBa_K3037000) as a vector were set. The BioBricks used were:

• A fusion protein of MBP and HRP (BBa_K3037008)
• This HRP but in the RFC10 standard (BBa_K1800002)
• This HRP adapted to the RFC25 standard (BBa_K3037007)

Then a culture of 100 mL E. coli pRARE T7 transformed with the different BioBricks was cultivated until the OD reached 0.5, then the culture was induced with 0.5 mM IPTG and 6 hours after that it was spun down. The pellet was stored at -80 degrees and left overnight. The next day the cells were lysed and the supernatant was taken to measure the protein concentration. The results were compared with the standard curve to calculate the concentration as shown in Figure 3.

Figure 3: Protein concentration of HRP measured in the cleared lysate of E. coli pRARE T7 carrying the expression backbone pOCC97 (BBa_K3037000) with different inserts. Expression was induced 0.5 mM IPTG

3) Protein Expression monitoring in SDS-PAGE:

A culture of 100 mL Escherichia coli pRARE T7 were transformed with the HRP BioBrick inside pOCC97 (BBa_K3037000), they were cultivated until the absorbance reached 0.5. Samples before induction were taken. Then the culture was induced with 0.5 mM IPTG,every 30 minutes samples were collected (5 samples), then 3 more each 1 hour. Before making the SDS-PAGE, the samples were adjusted to absorbance of 0.5 to have the same amount of cells in each lane. By doing so, the increase of a specific protein can be observed. The results show the increase of the concentration of the protein in time (Figure 4).

Figure 4: SDS-PAGE of the expression of HRP (BBa_K3037007) in the vector pOCC97 (BBa_K3037000). Upper anotations showing the different times of induction in minutes. The protein band is marked with an arrow

As it can be seen in the SDS-PAGE, the protein of interest is increasing over time from the point of induction onwards. HRP is marked with a black arrow pointing left.

4) Activity assay of the substrate conversion (TMB) compared to BBa_K1800002

The conversion of transparent TMB substrate to blue reaction product was monitored at 370 nm over half an hour with absorption measurements every 60 seconds for the HRP adapted to the RFC10 standard (BBa_K1800002) and to the RFC25 (BBa_K3037007) (Figure 5)

Figure 5: Activity assay of HRP in different iGEM standards expressed in pOCC97 (BBa_K3037000) monitored at 370 nm measuring every 60 seconds.

As it can be seen in the graph, the activity of this BioBrick and the original one from which it was adapted from are not different. This was our expected result, since the sequences are the same and only the prefix and suffix were changed from RCF10 to RCF25.

References

Krainer, F. W., & Glieder, A. (2015). An updated view on horseradish peroxidases: recombinant production and biotechnological applications. Applied microbiology and biotechnology, 99(4), 1611-1625.

Lamport, D.T.A. 1986. Roles for peroxidase in cell wall genesis, p. 199-208. In: H. Greppin, C. Penel, and T. Gasper (eds.). Molecular and physiological aspects of plant peroxidase. Univ. of Geneva Press, Switzerland.

Lamport, D.T.A. 1986. Roles for peroxidase in cell wall genesis, p. 199-208. In: H. Greppin, C. Penel, and T. Gasper (eds.). Goldberg, R., A. Imberty, M. Liberman, and R. Prat. 1986. Relationships between peroxidase activities and cell wall plasticity, p. 209-220. In: H. Greppin, C. Penel, and T. Gasper (eds.). Molecular and physiological aspects of plant peroxidase. Univ. of Geneva Press, Switzerland.

Sequence and Features