0 Introduction
Producing reliable cultivation data can be fairly difficult, time consuming and pricey.
We decided to adress this problem by creating OPEN PBR, a modular cultivation setup for photosynthetic organisms with up to 9 separate cultivation vessels.
While gathering knowledge with our efforts in Human Practices, we quickly recognized the need for a platform that enables the user to vary many parameters during cultivation of microalgae while still being affordable and capable of generating reproducible data.
The OPEN PBR fulfills these demands by providing a modular setup for cultivation of algae in turbido- or chemostat mode. With all components defined an openly available, it remains highly modular while using standardized vessels to provide reproducibility.
By using an existing model of general equations describing a chemostat with light as limiting substrate, we were able to pin down necessary functions and devices for performing them. For easy assembly of the device, files for lasercutting, a list of components and where to order them and an assembly guideline are provided.
1 Design of the open PBR
Design Overview
Type IIS restriction enzymes cut outside their recognition sites, making them useful in this cloning method for consecutive assembly of fragments. Through the restriction, overhangs are formed which allow the fusion of said genetic fragments to complementary overhangs of the syntax and thereby determine the order of each in a transcriptional unit (Figure 1). These fusion sites allow for the assembly of several fragments in the right order in just one cloning reaction. The used MoClo-kit offers ten different options for the positioning inside a L1 plasmid which are defined by the parts’ functions.
Fig. 1. Universal MoClo fusion sites.
Within the MoClo syntax, there are three different cloning vectors, level 0, 1 and 2 (referred to as “L0”, “L1” and “L2”, respectively). L0 vectors carry one basic genetic fragment or part, L1 vectors are assembled fragments creating a transcriptional unit and L2 are multigenic constructs. Construction of an L0-part is done by flanking a gene of interest with the specific fusion site and the recognition site of BpiI by a PCR reaction. Upon digestion by BpiI it can be inserted into a previously digested L0-backbone. To then clone it into a L1-backbone, it is digested by BsaI, revealing the fusion sites for its assembly in a transcription unit. Lastly, a fusion of several transcription units (L1) into a L2 multigenic device is possible with the MoClo syntax.
As part of our contribution to a toolkit usable by future iGEM teams we design and construct not only the parts we intend to use on our goal of PET-degradation but several more, a L0-backbone and L1-backbone.
To ensure that all parts were designed correctly we cloned the PCR fragments into a L0 vector. To this end, we used a self-modified version of a L0 backbone containing RFP. After ligation, we transformed the L0 plasmids containing the parts into Escherichia coli and checked for white transformants. Only genes with the correct fusion and restriction sites could be inserted into the L0 backbone resulting in growth of white colonies, since the RFP gene was interrupted. For further verification, we checked the parts by DNA sequencing. We used the same control mechanisms for L1 assembly constructs.
Fig. 2. Overview of the Golden Gate cloning strategy.
2 Overview of single components
Single compos intro
Fig. 3. Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid.
-
sensors
illumination
gas mixing
pumps
cultivation vessel
casing
2 Overview of single components
-
Github Project
Tools
PDF Instructions
3 Assembly instructions
