FB 17 - Biologie, Karl-von-Frisch-Straße 8, 35043 MarburgWeb: http://www.uni-marburg.de/fb17

LOEWE-Zentrum für Synthetische Mikrobiologie, Hans-Meerwein-Straße,Mehrzweckgebäude Raum 06 C 18, 35032 MarburgWeb: http://www.synmikro.com

Safety: Red or Dead

In order to prevent a survival of PHAECTORY cells in nature we develo-ped a two-stage security system triggered by blue light. In the first stage a LITE1 module (light-inducible transcriptional effector) is under the control of a weak, constitutive promoter resulting in a cons-tant low level of LITE1 in the cell. LITE consists of a transcription activator-like effector (TALE) fused to a blue light sensitive cryptochro-me 2 protein (CRY2). The TALE1 and the attached CRY2 bind to the TALE1 binding site upstream of the LITE2 module. LITE1 stays inactive until illumination with blue light. When blue light reaches CRY2 confor-mational changes happen that activate the LITE1 module resulting in the expression of LITE2. LITE2 in turn binds to the TALE2 binding site lea-ding to the expression of Protease K and DNaseI, which finally leads to cell death.

Nature PHAECTORY

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LITE2: CRY2TALE2

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For the technical realization we suggest a blue light filter for PHAECTORY. In the unlikely event of an algae outbreak, the existing blue spectrum of the sunlight will lead to the immediate cell death.

Directing a red fluorescent protein to the membrane

An important aspect in synthetic biology is the cellular com-partmentalization of complex enzyme reactions. Therefore, it would be great to establish parts that allow the recruit-ment of synthetic components to cellular membranes.

Due to this, we decided to establish membrane scaffolds, which are known as membrane targeting sequences (MTS). These sequences form amphipathic helices, which autono-mously bind to membranes often with specificity to anionic phospholipids. To challenge that idea, we fused the reporter RFP (red fluorescent protein) from the registry with a C-terminal MTS. The localization of RFP-MTS was shown in Escherichia coli where it showed a slight preference for the poles. Taken together, we were able to create an additional BioBrick for our toolbox and to improve the in the registry existing RFP.

Secretion

We challenged PHAECTORY as a green system for the pro-duction of antibodies which are directly secreted into the pure surrounding medium. The secretion of the antibodies is mediated via the regulated secretory pathway.

The genes for the Hepatitis B antibody produced in PHAECTORY are encoded by the nuclear genome where transcription takes place. The produced messenger RNA contains an amino terminal signal peptide, which directs the protein into the endoplasmic reticulum (ER). The antibodies enter the ER as nascent proteins and are then folded and N-glycosylated in the lumen. Afterwards the antibodies are transported to the Golgi apparatus via COP II (coat protein complex) vesicles where further posttranslati-onal modifications like the O-glycosylation are executed.

Thereafter the antibodies are packaged into secretory vesic-les, which are directed to the cytoplasmic membrane where they fuse with the membrane leading to the release of the antibodies. However, the exact mechanism of the secretory pathway in plants is not yet well characterized.

Characterizing a new light-inducible promoter

Because plants and algae use sunlight as their primary energy source, they had to develop promo-ters, which respond to light. We challenged the idea whether these light-inducible promoters would be suitable for regulating expression of target genes. Therefore we studied the strength of the light-inducible promoter fcpB, which was fused to the reporter eGFP, by radiating the cells with dif-ferent transmission wavelengths (i.e. green (572 nm), blue (467 nm), red (672 nm), white light and darkness). The amount of eGFP was quantified by Western blot analysis. For the evaluation of the Western blot normalization against optical density (OD) was done.

It is clearly evident that light of any wavelength leads to the activation of the light-inducible promo-ter. There is almost no visible difference in the amount of eGFP in the cells irradiated by blue, green and white light. In contrast, red light leads to more than 1.5 times the amount of eGFP.

PromotereGFP

PHAECTORY

To challenge PHAECTORY for the efficient antibody production and secretion we constructed ex-pression vectors with genes coding for the Hepatitis B antibody and transfected them into the algae Phaeodactylum tricornutum. The genes were placed under the control of nitrate-induciable promoters. Five different clones of PHAECTORY were grown to an optical density of 0.4 (OD600) in a nitrate-containing medium. To see how much Hepatitis B antibody was secreted from the algae into the surrounding medium, both intact cells (pellet) and supernatants were analyzed by Western blot.

The Western blot analysis shows that huge amounts of Hepatitis B antibodies were secreted into the medium. The positive signal in the cell pellet shows that antibody production by PHAECTORY was still in progress. Taken together, the amount of Hepatitis B antibodies was significantly higher in the supernatant then in the cell pellet. This is the ‘proof of concept’ that PHAECTORY has not only the ability to produce high-value proteins (e.g. antibodies), but also secrets them in huge amounts into the surrounding medium.

Introduction

The use of proteins in medical treatment and diagnosis is steadily increasing. Many of these proteins (e.g. antibodies) have a complex biological structure, which complicates their production. Also, these proteins need to be highly pure. Therefore, a major challenge is the development of systems that produce complex proteins with high purity - best at low costs! The sunlight-driven microalgae Phaeodactylum tricornutum has been used for the production of complex proteins. A major advantage of the algae is its ability to secrete proteins directly into the medium. This feature would greatly simplify purification of recombinant proteins, and lower production costs! Here we take advantage of the excellent secretion abilities of P. tricornutum, and make this organism accessible to synthetic biology and the iGEM competition.

Furthermore, we use a light-inducible promoter which we have quantitatively characterized in order to control protein expression in PHAECTORY. Also, we introduced a transferable element to the registry, which allows autonomous targeting of proteins to the inner surface of cell membranes.

PHAECTORA n t i b o d i e s p r o d u c e d b y s u n l i g h t

iGEMMarburg

Picture: A. Grubner, University of Konstanz