Design

On the biological side, the idea was to increase expression of a THB1 (truncated hemoglobin 1) gene in cells. Indeed, heme-associated iron is the best absorbed in the intestine. Therefore, the controlled overexpression of a heme protein capable of binding iron was the challenge of our project.
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We decided to express the THB1 transgene in a Nit2- strain (UVM4). The THB1 protein is regulated by the transcription factor Nit2, which is the major transcription factor in nitrate assimilation. By using a Nit2- strain, THB1 expression naturally present in C.reinhardtii is prevented. We can therefore have a completely controlled expression of the transgene. 

In order to maximize this control, the use of insulator sequences was chosen. Insulator sequences are found in certain eukaryotes, and make it possible to protect a gene or a group of genes from the effects produced by neighboring regulatory sequences. During the stable transformation, endogenous transcriptional enhancers can interact with promoters in artificial systems, or genomic heterochromatin can spread, which leads to an obstacle in the expression. In order to obtain a stable expression, the use of insulator sequences framing our gene system is therefore essential.

However, the existence of insulator sequences in C.reinhardtii has never been demonstrated. Several solutions were therefore available to us. 

First of all, the gypsy insulator, which is the most characterized enhancer-blocking insulator. It is found within the 5’-untranslated region of the gypsy retrotransposon from Drosophila (∼350bp). This insulator sequence works with zinc finger proteins Su(Hw) (Suppressor of Hairy-Wing), which recruit additional factors in order to form the “insulator body”. The gypsy insulator and other non-plant insulators have been shown to be functional in plants, such as transgenic Arabidopsis thaliana. This suggests the conservation of components involved in insulator activity in plants (ex: proteins). As C.reinhardtii is one of the greatest models for superior plant, insulator sequences such as the gypsy one could work in this organism. 

The second solution was to find insulator sequences directly present in plants as C.reinhardtii is an eukaryotic photosynthetic organism. One of the plant insulator candidates is the gypsy-like sequence (2258bp) found in the Arabidopsis thaliana genome.

‍Finally, to complete the control of expression, we chose to use an inducible promoter to express THB1. Indeed, we want to grow our algae in bioreactors, and in order to get rid of the burden that our transgene might impose on C.reinhardtii's metabolism that could impact its growth, we wanted to be able to choose when to induce the expression. Moreover, we wanted to grow our algae in the dark, so that there would be less chlorophyll, which has an impact on the bad taste of algae. Therefore, we choose to use a light inducible promoter, such as the high light-inducible Dunaliella LIP promoter  which is easy to use and does not require any addition to the medium.

Our final plasmid should therefore be composed of an origin of replication, the insulator sequences, the THB1 gene and an antibiotic resistance gene in order to facilitate the selection.

Build

In order to build our plasmids we relied on the MoClo kit for C.reinhardtii. MoClo (Modular Cloning) is a standardized method based on the use of building blocks and Golden Gate cloning. Each standardized gene part (level 0) is designed and cloned for a specific position in the transcription unit (level 1). Several transcription units can be combined (level M) to allow the expression of several genes at the same time. The MoClo kit is therefore perfectly scalable, with easy exchange of parts, if designed for the same position, by adding specific sites.

When we first tried to construct the plasmid, we wanted to use the Atgypsy sequence as the insulator sequence. However, this sequence is very long (2258bp) and isolating it from the Arabidopsis thaliana genome or producing it via our sponsors was not easy. We therefore decided to express the gypsy sequence as well as its Su(Hw) gene sequence in our construction. Each standardized gene part is therefore manufactured or being manufactured to obtain our final plasmid.

Test

Before testing our complete design, we first had to test and prove that the gypsy insulator sequence together with its Su(Hw) protein fulfilled its function in the model organism C.reinhardtii.
Two plasmids have been designed and are under construction. These are a test plasmid (pM-insulator) and a control plasmid (pM-control). The test plasmid consists of the insulator sequences, a gene encoding a fluorescence protein (mVenus) under the control of the constitutive promoter (pPSAD), and the gene encoding the Su(Hw) protein. While the control plasmid has only the fluorescence protein gene under the control of the constitutive promoter.
After transformation of the UVM4(Nit2-) strains with the two plasmids and culture in multi-well plates, the test is performed using a plate reader. The objective is to measure the fluorescence intensity of mVenus in colonies with the test plasmid and those with the control plasmid.
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The proof of concept is that the fluorescence intensity varies for colonies with the control plasmid and is constant for colonies with the test plasmid. Indeed, as the gypsy sequences isolate the transgene from the genomic context, the expression of the fluorescent protein should not vary.