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Design

As previously explained on our Description page it is our aim to establish C. reinhardtii in the iGEM competition. To reach this goal we created a toolkit, "Chlamy-HUB Collection", of various functional parts and multi-use constructs that future iGEM teams can use and optimize.

So, what is our focus?

1. C. reinhardtii at iGEM

2. PET-Degradation

3. Bioreactor

1. Establishing Chlamy in the iGEM competition

• 1.1 Golden Gate Modular Cloning for Chlamydomonas reinhardtii

• 1.1.1 Selection cassette construction

• 1.2 Protein expression analysis - testing our transgenic proteins

  • 1.2.1 Secretion signal

  • 1.2.2 PtxD - phosphite decarboxylase

  • 1.2.3 Promoter comparison tests

  • 1.2.4 Cas9/sgRNA-mediated site-directed mutagenesis

• 1.3 Modeling photoautotrophic growth of Chlamy

1.1 Golden Gate Modular Cloning for Chlamydomonas reinhardtii

To synthesize and assemble the genetic elements for the Chlamy-HUB Collection, we applied the Type IIS “Golden Gate” cloning strategy (Engler et al., 2008). We used the Modular Cloning (MoClo) toolkit optimized for C. reinhardtii (Crozet et al., 2018), which follows the MoClo syntax of the plant synthetic biology community (Patron et al., 2015).

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. 12 fusion sites (Patron, 2015) for the seamless fusion of different level 0 parts. In general, the fusion sites are grouped into 5’ untranslated regions including promoter sequences (grey), the translated coding sequence CDS (blue) followed by 3’ UTRs ending with a terminator (orange). Within these types, various parts (e.g. three different coding sequences) can be designed and combined into one transcription unit when cloned together into a L1 vector. The bold ATG within the B3 fusion site sets the transcription start.

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. Multiple basic genetic elements on a level 0 vector (Phytobricks) can be assembled to a full transcription unit on a level 1 vector. Specific fusion sites (shown in grey) and BpiI recognition sites are added to new genetic elements via PCR. To build a L0 construct the Type IIS restriction enzyme BpiI digests the PCR fragment and the L0 vector. The L0 vector, in turn, contains the recognition site for BsaI. Digestion with BsaI and ligation of several L0 parts with a L1 backbone leads to a L1 transcriptional unit. The different L1 modules of choice can then be assembled via BpiI into the final L2 construct in which no recognition sites for type IIS restriction enzymes are left.

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1.1.1 Construction of a selection cassette

Standard vectors for MoClo cloning are equipped with antibiotic resistance cassettes for cloning in E. coli. But since this work is focused on the expression of PET degrading enzymes by C. reinhardtii, the final transformable devices need to provide a selection advantage for these algae.

Overview of the construction of selection cassettes.

Pierre Crozet and his team created a MoClo toolkit adapted for the model organism C. reinhardtii with 119 openly distributed genetic parts (Crozet et al., 2018) including resistance cassettes. However, these resistance cassettes are not located on any level 1(L1) plasmid backbone. Consequently, an additional cloning step is required to assemble a L2 device, since a single L1 construct cannot be transformed without a resistance cassette. To simplify this cloning process, we design a resistance cassette specifically for C. reinhardtii to serve as a plasmid component for the transformation into Chlamydomonas reinhardtii.

To build a selection cassette using the Golden Gate cloning standard, each part required for a selection cassette (promoter, linker, selection marker and terminator) must be cloned into the L0 backbone. The “selection marker” can be any antibiotic resistance for C. reinhardtii. Golden Gate cloning using BsaI can assemble the parts into a L1 backbone which results in an antibiotic resistance transcriptional unit. Using primers amplifying the selection cassette on a L1 MoClo backbone attaching the restriction sites compared to restriction sites on the “new” L1a,b,c vectors. Attaching BamHI and XhoI sites gives rise to a PCR product which is linkable with the new constructed L1 plasmids when digested by BamHI and XhoI. The resulting plasmids can then represent level 1 Golden Gate cloning vectors specified for C. reinhardtii. The picture shows an example to produce a selection cassette containing a hygromycin resistance linkable to a new L1c plasmid.

Fig. 3 - Cloning process to insert a selection cassette into the L1c-RFP_ampR/Ori plasmid. The selection transcription unit (PsaD-B2Linker-hygR-Rbcs2) inside the pICH47732 backbone can be amplified with primers containing restriction sites for BamHI and XhoI to receive a PCR product linkable with the new constructed L1 plasmid L1c-RFP_ampR/Ori when digested with BamHI and XhoI. The resulting plasmid L1c-RFP-HygR represents a level 1 Golden Gate cloning vector specified for C. reinhardtii.

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1.2 Expression analysis - testing our transgenic proteins

The composite parts we created contained different markers, antibiotic resistances or the phosphite marker PtxD, that allow for selection of transformed algae clones. Depending on their intended utility for C. reinhardtii, each of our constructs is screened in a different way to prove their intended function.

Fig 4 - Visualization of the YFP selection process of transformation clones. After growing on a selective media, the clones are screened for expression of the transformed transgenes. The screening methods are dependent on the intended function of each construct.

Once constructed, the designed plasmids are transformed into the SAG32-11b and UVM4 strains of C. reinhardtii and tested for expression. As a transformation method we chose electroporation, during which two different electric fields are applied to the algal cell. The first, a high voltage pulse applied for relatively short time causes the membrane to form pores and the second, of a low voltage for relatively long time transfers the DNA into the cell. We co-transform each of our constructs containing the transcriptional unit (L1 constructs) with a plasmid conferring antibiotic resistance (paromomycin or hygromycin, in our case).

After transformation, the algae has to be tested for successful uptake of the plasmids. If the clones are able to grow on TAP-agar plates containing antibiotics, the resistance plasmid was transformed and is proven to work accordingly. Subsequently, the clones have to be screened for the presence of the genetic constructs. A colony PCR is used to amplify the constructs. If the respective bands show up, the clones are continuously cultivated under thriving expression.

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1.2.1 Testing secretion signals

As many enzymes function outside of its host organism, we want to recreate the secretion of heterologous expressed proteins in C. reinhardtii. Secretion is possible due to a signal sequence attached to the protein at its 3’UTR that leads it to the secretory pathway.

We take native signals from arylsulphatase 1 (ARS1) and a putative signal sequence of gametolysin (GLE) (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012) and build L0 modules with them. As the yield of heterologous expressed proteins fused to these secretion signals was previously described as relatively low, we additionally designed a 5’UTR glycomodule (SP₂₀) as a L0 part which also enhances the stability of the attached protein when secreted (Ramos-Martinez, Fimognari, & Sakuragi, 2017; Rasala et al., 2012).

In order to test these secretion signals, we tagged them with a YFP protein, which can be observed under a fluorescence microscope (excitation peak at 480 nm and emission peak at 528 nm). If secreted, the medium outside the cells is expected to emit fluorescence as opposed to the medium surrounding wild type algae.

Fig. 5 - Secretion of transgene proteins into the media by C. reinhardtii. Secretion tags in form of signalling peptides added to expressed proteins in the ribosomes. These lead to secretion of said protein through exporters, a process during which the secretion tag is lost.

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1.2.2 PtxD - Phosphite Oxidoreductase

Algae depend on phosphate as their sole phosphorus source. Phosphite, the reduced form of phosphate however, competes with phosphate for transport proteins and therefore is harmful to plant growth.

Fig. 6 - Growth comparison of wildtype C. reinhardtii and PtxD-expressing mutant in phosphite media. In the case of the wildtype algae, growth is inhibited because the metabolization of phosphite is not possible and these strains therefore lack phosphorous. The PtxD-containing strains however are able to import and successfully integrate phosphite into their phosphorous metabolism.

The phosphite oxidoreductase from Pseudomonas stutzeri WM88 (PtxD) oxidizes phosphite to phosphate using NAD⁺ as a co-substrate (Loera-Quezada et al., 2016; White & Metcalf, 2007). We provide our C. reinhardtii strains with the PtxD enzyme to increase its competitiveness against other microorganisms in culture. This way, we enable it to degrade phosphite, presumably as only organism in our culture, avoiding contamination.

We observe the growth of our engineered C. reinhardtii in media containing phosphite, phosphate or phosphorus to prove the enzyme activity. In the phosphite-supplied media, we expect to observe growth of the strains expressing PtxD and inhibition of wild type algae due to a lack of phosphorus.

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1.2.3 Promoter comparison tests

Knowing the conditions under which a transgene is transcribed optimally can be very useful. Therefore, we decided to test two of our promoters even further: the PsaD-Promoter from subunit II of Photosystem I and the AR promoter which is a fusion promoter of RbcS2- and HSP70-promoters as well as of the RbcS2 Intron 1.

To test if they behave as expected, we designed constructs making it possible to directly compare them by expressing YFP as fluorescent marker protein under the regulation of both promoters. Using a plate reader and fluorescence microscope made comparing numerous AR- and PsaD-constructs very easily. We also planned to compare expression rates of constructs containing the PsaD promoter with and without introns. PsaD with introns was designed taking the AR promoter as a model. The AR promoter is composed of the HSP70 promoter fused to the RbcS2 intron. We followed a similar approach by fusing the RbcS2 intron to the end of the PsaD promoter, hoping to achieve a higher expression yield. Unfortunately, we were not able to compare expression driven by these three variants of promoters in the wet lab.

Fig. 7 - Comparing the efficiency of PsaD promoter with and without intron. The presence of YFP protein expressed under each promoter variant is assessed by fluorescence microscopy first. Then, the amount of YFP is determined by measuring with a plate reader using a calibration curve of known protein amount-fluorescence measurements.

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1.2.4 Cas9/sgRNA-mediated site-directed mutagenesis

When establishing Chlamydomonas algae as a viable chassis for expression of synthetic constructs, one must consider how to ramp up the expression of such constructs. One solution might be to guide the insertion into a certain genome locus. As has been proven before, gene insertions into the nuclear genome of C. reinhardtii can be achieved by transformation of a recombinant Cas9 protein, combined with a guide RNA (gRNA) (Kelterborn et al., unpublished data).

Target recognition inside the nuclear genome of C. reinhardtii relies on the presence of protospacers and protospacer-adjacent motifs (PAM) on the target DNA. A gRNA, which is designed to match specific regions on the genome and bind to them (Mali et al., 2013) is used to direct the Cas9 endonuclease. Upon binding to the target region, a cut of Cas9 creates a double-stranded break (DSB) with blunt ends. Into the DSB artificial DNA could be inserted through DNA repair-mechanisms. We wanted to find out if there were regions in the genome of C. reinhardtii that displayed an increased protein expression. To this end, we designed sgRNA complementary to three different genome regions.

The first locus is the SNRK locus. Since Kelterborn et al. had implemented a straight-forward screening process, successful cleavages could easily be identified. We want to use these loci mainly as a comparison to different locus insertions, since we do not expect high expression. As we are using the Promoter of the Photosystem Subunit II, we want to check if we are able to increase the expression by inserting our DNA Fragments at the end of the PsaD gene. Strenkert et al. (2019) have shown that a light dependent increase of PsaD expression occurs. When grown under synchronized light conditions a higher protein yield may be achieved.

Ribulose-1,5-bisphosphat-carboxylase (RuBisCO) is often referred to as the most abundant soluble protein. Carrying the function of carbon dioxide fixation it is present in all plants and phototrophic organisms. Due to this frequent appearance, we want to see if insertions inside the RuBisCO locus lead to higher expression. By targeting this locus for the insertion of our constructs, we hope to increase the protein yield, while characterizing the expression pattern of C. reinhardtii to ease the further use for the SynBio community.

During the process of electroporation transgenic DNA is inserted randomly into the genome of C. reinhardtii. This opens up the possibility that a high expression locus might be found by coincidence during our screening process. This locus could then be considered for further transformations to yield a high protein expression, useful in all genetic engineering projects.

To identify such a locus we try to amplify this genomic region with a restriction enzyme site-directed amplification PCR (RESDA-PCR). This polymerase chain reaction uses primers that are designed in a way that they anneal at restriction enzyme recognition sites of the enzymes AluI, SacII, PstI or TaqI. These sites are widely spread inside the genome. This increases the probability that the transgene DNA is inserted next to one of these sites, so that a PCR-product containing the genome sequences might be amplified and sequenced.

Fig. 8 Crisp Cas9 and RESDA-PCR Inserting genes through Cas9 and verifying their position through RESDA-PCR

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1.3 Modeling photoautotrophic growth of Chlamy

In a first step to facilitate the ‚Design - Build - Test - Learn‘ cycle, we wanted to create a model that has the aim to give an overview of metabolic processes, genes and other parameters necessary for photosynthetic growth.

Fig. 9: Overview of the components of a growth model. Each component of our analysis is derived from the function it has in Chlamy's growth. Adapting the parameters can give insight into the best way of cultivation for the optimal growth conditions, for example, in a bioreactor.

We use the syntax defined in the Constraint Based Reconstruction Analysis (COBRA) Toolbox for Python (Ebrahim et al., 2013), already existing metabolic reconstructions of Chlamy (Imam et al., 2015, Kliphuis et al., 2011), the fully sequenced genome (Merchant et al., 2007 ) and a recent -omics dataset (Strenkert et al., 2019) to define the components of the model. In a second step we combine these components in such a way that we are able to assess how they work together to give rise to growth of a Chlamy culture. The model should also provide the synthetic biologist with information useful for performing specific tasks in C. reinhardtii.

Unfortunately, we could not finish the model because there was not enough time. We still were able to use knowledge gathered along the way, though.

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2. Working on PET-Degradation as a proof-of-concept

• 2.1 Modeling PET degradation by C. reinhardtii using an optimized PETase

• 2.2 Transformation of PETase and MHETase into C. reinhardtii

• 2.3 Testing the toxicity of TPA and EG for C. reinhardtii

2.1 Modeling PET degradation by C. reinhardtii using an optimized PETase

A C. reinhardtii which expresses and secretes the enzymes PETase and MHETase could pose as a solution for the problem of micro-plastic polluted water. Nevertheless, the viability of PET-degradation by C. reinhardtii at a larger scale is yet unknown.

To assess the efficiency of PET-degradation by C. reinhardtii, a model of the expression, secretion and kinetics of the enzymes PETase and MHETase in C. reinhardtii was designed. The model can be seen here. The goal of the model is to simulate the degradation of PET while taking into account the parameters of enzyme kinetics, expression rate, secretion rate and cultivation density. By varying the parameters, an approximation on PET degradation under various conditions can be made to examine what the appropriate parameters are for an optimal PET-degradation. To achieve this, the kinetics of MHETase and the optimized PETase (I179F) were taken from literature (Palm et al., 2019, Ma et al., 2019).

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2.2 Transformation of PETase and MHETase into C. reinhardtii

Expressing and secreting the enzymes PETase and MHETase with C. reinhardtii could pose a solution for the problem of micro-plastic polluted water. Nevertheless, the viability of PET-degradation by C. reinhardtii at a larger scale is yet unknown.

Plastic degradation as intended by our project is to take place in the media of C. reinhardtii after expression and secretion of both the PETase and MHETase enzymes by the algae. To test the expression of PETase different constructs need to be implemented. We apply YFP as a fluorescent tag to optically screen our transformed clones for expression of the plasmid and therefore, PETase-production.

Our other tag, the 3xHA-tag was intended for purification of the enzyme out of the C. reinhardtii cells. As secretion signals we chose the arylsulphatase 1 (ARS) and gametolysin (GLE) secretion signals, which were compared to each other. The serine-proline glycomodule (SP_₂₀) is a secretion enhancer. While not only important for our goal to degrade plastics but also crucial in developing a toolkit for multi-purpose use, we test the following constructs:

• PETase with secretion signal GLE and fluorescent tag YFP
  
  
• PETase with secretion signal ARS, glycomodule SP_₂₀ and YFP
  
  
• PETase with HA purification tag and ARS
  
  

First, the clones of each transformation are tested for the uptake of the plasmids via colony PCR. Then, if bands of the expected length appear, the respective clones are continuously cultivated and algae cell suspensions are tested for fluorescence signals via fluorescence microscopy. If the secretion signals work, the medium around the cells emits fluorescence. This screening step is the most time-consuming. Once clones displaying fluorescence are detected, we try measuring the comparative fluorescences of the engineered clone and wildtype algae in a plate-reader. Constructs coding for the expression of MHETase are MHETase with YFP as a fluorescent tag, enabling screening for transformants and with an HA-tag for purification.

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2.3 Testing the toxicity of TPA and EG for C. reinhardtii

As it is our goal to grow C. reinhardtii in a bioreactor in which it secretes PETase and MHETase we need to understand how it can deal with the produced degradation products terephthalic acid (TPA) and ethylene glycol (EG).

Within this framework we measured the growth rates of several C. reinhardtii strains in a series of experiments.

With the help of the Multi Cultivator MC 1000 we can test four different C. reinhardtii strains on these reagents to find out which one is the most suitable for further experiments and for transformation.

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3. Designing and building a bioreactor

Along the way of performing our experiments and making our parts, we recognized that there is one big bottleneck in growing not only C. reinhardtii but also other photosynthetic organisms: lack of an affordable and reproducible cultivation setup in a laboratory scale.

We therefore decided to address this problem by designing and constructing an open source platform that is capable of growing phototrophic organisms in turbidostat mode, not only monitoring growth but also making it possible to adjust environmental conditions.

To help us in the process of design, we asked several sources with experience and knowledge in algae cultivation on different scales to get a first overview of problems and demands of cultivation (Joerg Ullmann, Gunnar Muehlstaedt, Ralf Steuer). With this help and knowledge gathered from modeling, we were able to pin down necessary functions our platform has to fulfill and therefore components it has to be built of. We always keep one function important to the Synbio community in the back of our head: The ability to perform many experiments and/or screening events at once. This means that a general demand for our setup is to be easily affordable, reproducible and manufacturable with as little effort as possible.

Taking these demands and information into account we decided on the following functions and components performing them: Different types of cultivation vessels with self built flat panel for higher density; Photometer to measure optical density; Regulation of air supply and mixing of the culture; Photon source e.g. LED; Temperature Measurement and control; Control of pumps in a Turbidostat setup As every single component gives rise to necessary tests for assessing its function (such as calibration of the photometer and pumps), details of experiments performed are depicted on the bioreactor subpage.

We will create a library of step by step tutorials showing how to build all of these different functions and make it possible and motivate other iGEM participants and scientists to engage in do it yourself (DIY) setups and electronics.

To perform our needed functions, we will build a small low-cost DIY photometer which can measure the optical density (OD) of C. reinhardtii at 680 nm or any other wavelength in the visible spectrum. The photometer is designed to not only work inside our cultivation setup, but can also be used as a stand-alone solution for OD measurement.

Given the possibility of using several different cultivation vessels, we designed our setup to hold standard Schott 45 flasks, cell culture flasks with 3D printed screw-tops and flat panels designed by ourselves, while being able to hold any other cultivation vessel with the appropriate dimensions.

Gas supply and mixing of a culture are important factors to maintain a constant cell growth and, therefore, have to be monitored closely. We tried to design a solution with a rotameter which is easy to control to achieve a high chance of reproducibility. It is also possible to measure and regulate the temperature of the cultivation vessel to obtain reliable data.

For temperature control, we decided to immerse our cultivation vessels in a water bath where temperature can be measured and held constant through a heating element.

Because in a photobioreactor setup the photon source is probably the most expensive and complicated part, we decided to build in a RGBW light source with an intensity of at least 300 µE to make light-dependent growth at high densities possible.

To be able to reproduce results from Flux Balance Analysis and perform other experiments which demand steady state, we wanted to include a turbidostat-function in our setup by installing two pumps that can hold OD of the culture constant. Removed culture can be used for transformation experiments or for screening.

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Results

This is the data we collected with our experimental set-up. Click here to see what we found out.

Experiments

If you are interested in our day-to-day lab protocols or standard workflows - our Experiments page lists all resources you might need.

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