Contribution

Building on prior iGEM work, we’re expanding the community’s toolkit for algae. Our team provides step-by-step instructions and 3D-printable files for a medium-sized bioreactor, plus genetic parts for precise knock-ins in the nuclear genome of Chlamydomonas reinhardtii. Together, these resources enable long-term, reliable expression at scale—so the next teams can go from bench sketch to glowing culture, fast.

Genetic Parts

Random genome integration, as described in the Chlamy Guide, is and will stay the quick-and-dirty version of genome integrations. For more sustainable, and usually stronger, gene expressions, precise knock-ins via the CRISPR/Cas system are used [1, 2]. We provide homology arms (500 bp on each side of the CRISPR locus) as well as the matching guide RNA sequence which have been used in literature [3].

Part Name	Description
BBa_25MSP6W5	Left homology arm (upstream of locus)
BBa_25625BCW	Right homology arm (downstream of locus)
BBa_25FOJWXS	Guide RNA

A three-part CRISPR/Cas toolkit is presented for nuclear transformation of Chlamydomonas reinhardtii, guided by established design principles and targeting the SNRK2.2 locus on chromosome 12. Disruption of this gene enables straightforward selection, as edited colonies develop a blue-green/indigo phenotype via aryl-sulfatase activity, when the medium is treated with chromogenic arylsulfatase substrate [5]. The design favors practical screening while keeping constructs manageable. Primer design and PCR confirmation proved difficult for this genomic region, yielding diffuse rather than distinct bands on agarose gels. Simulations indicate elevated self-complementarity at primer binding sites flanking the homology region. Targeted refinements to primer design and construct architecture may enhance the efficiency and reliability of this toolkit.

Figure 1: An illustration of the mechanism of the selection marker.

The HDR arms can be deployed with the complete expression cassette described in the Engineering section. A suitable antibiotic resistance marker may be selected according to experimental requirements. AlphaFold simulations indicate that the mVenus fluorescent reporter, commonly used in C. reinhardtii transformation, can be fused to the TFD protein without disrupting folding or function. Alternatively, expression levels may be assessed via protein purification and quantification using a C-terminal Strep-tag. When combined with the phenotypic screening described above, these three layers of selection enable precise determination of transformation efficiency and expression levels, while greatly facilitating troubleshooting.

Bioreactor Material

We provide a comprehensive Guide (see Hardware) with a shopping list and instructions on how to build a Bioreactor similar to ours. The basic model was pitched and inspired by Fabian Abiusi[4].

Here are some approximate specifications of the reactor.

Information	Value
Volume medium	15 + liters. Our specimen has a volume of 18 L (and is filled up to 16 L)
Biomass Production	Approximately 4.5 g per day with Chlamydomonas reinhardtii (corresponds to 0.28 g/L/day) Productivity measured under autotrophic conditions; values will differ under mixo/hetero, other strains, or light regimes. Note: these specs are approximations based on literature[4].
Advantages	Automatic regulation of temperature via temperature probe and a controlled fan for air cooling.
Disadvantages	Cultures should not be kept longer than 2 days after full colonalization of the reactor, according to Abiusi. After, the risk of contamination increases as the system is open.