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 with pOOC97 (BBa_K3037000) in E. coli pRARE T7

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

3) Protein Expression monitoring in SDS-PAGE

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

Experiments in Detail

1) Growth curve of expression with pOOC97 (BBa_K3037000) in E. coli pRARE T7

The HRP was expressed using the plasmid pOOC97 as a backbone (BBa_K3037000)

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

For this the development of the culture 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 growing of the bacteria is not affected by the expression of the protein. Escherichia coli show a the normal growth behaviour as expected in a batch culture (Figure 1).

Figure 1: Growth curve of expression of HRP. Vector pOCC97 (BBa_K3037000). Induction with 1mM IPTG

2) Determination of the total protein amount 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: Growth curves of E. coli strains carrying our expression backbone with different inserts. Expression was induced after 165 min of incubation with 1 mM IPTG

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 100mL E. coli pRARE T7 transformed with the different BioBricks was cultivated until the OD reach 0.5, then the culture was induced with 0.5 mM IPTG and 6 hours after that it was spin down. The pellet was frezeed at -80 degrees and leaved overnight. The next day the cells were lysate and the supernatand was taken for measure the protein concentration. The results were compared with the standard curve to calculate the concentration as showed in the 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 transformed with the HRP BioBrick inside pOOC97 (BBa_K3037000), they were cultivated until the OD600 reach 0.5. Samples before induction were taken. Then the culuture was induced with 0.5 mM IPTG, samples were taken each 30 minutes (5 samples), then 3 more each 1 hour. Before making the SDS-PAGE, the samples were adjusted to OD of 0.5 to have the same amount of cells in each lane. This way the increase in one 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 can be seen in the SDS-PAGE the protein of interest is incresing 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 measurement 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

As can be seen in the graph, the activity of this BioBrick and the original one that it was adapted from are not different. This was to be expected, 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