Team:Sorbonne U Paris/Results

Building plasmids

All plasmids were constructed using the Modular Cloning (MoClo) method 1 adapted to Chlamydomonas Reinhardtii 2. This method, based on Golden-Gate assembly 3, allows first the assembly of parts such as promoters, coding sequences, signal peptides, etc. into a transcriptional unit in only one reaction and then, the assembly of full transcriptional units into multi-gene constructs again in a single reaction. Using this method, we were able to construct without difficulty all our plasmids.

HiBiT characterization

We aimed to produce plasmids that would be used to transform C. Reinhardtii in order to characterize the HiBiT tag in this organism, as well as adding a new part to the MoClo kit for this alga.

To construct plasmids expressing the HiBiT tag, we ordered two oligonucleotides for the two positions we wanted to use our tag, in N-terminal or C-terminal. We let them hybridize, and then start the reaction to build level 0 plasmids. To be sure that the initial oligonucleotide sequence was correct, we’ve sequenced all the plasmids obtained. Thus, we obtained level 0 plasmids with the HiBiT tag in N-terminal or C-terminal position.
Using parts of the MoClo kit provided by Pierre Crozet, we successfully constructed the level 1 plasmids build to express either GFP tagged with the HiBiT sequence in N-terminal position (pCM1-1) or C-terminal position (pCM1-2), GFP tagged with NanoLuc in N-terminal position (pCM1-4) or C-terminal position (pCM1-5) or GFP without any tag (pCM1-3). To confirm the presence of the transcriptional units in the plasmid, we digested the plasmids with Bbs-I, and performed an electrophorese on agarose gel. Doing so we were able to identify clones containing the transcriptional units.

We did not sequence our plasmids to ensure that they all contain the full transcriptional unit with right sequence because first, the Golden Gate assembly does not allow the circularization of a plasmid not containing all parts; and second, we did not use PCR to amplify DNA or any other process involving the synthesis of DNA, other than the replication of plasmids by the bacteria machinery, thus avoiding replication error leading to a modification of the DNA sequence.

In order to select algae cells transformed with our DNA, we needed to add to our constructions a resistance gene to an antibiotic. To build multigenic constructions we used again the MoClo assembly method to make level M plasmids, with two transcriptional units: one enabling the expression of the tagged GFP and the other allowing the cells containing the plasmids to resist to paromomycin. Using again the digestion/ligation reaction, we digested with Bbs-I the level 1 construct we obtained before as well as a level 1 construct enabling the expression of the paromomycin resistance gene.
Thus, we obtained constructs enabling the expression of GFP tagged with the HiBiT sequence in N-terminal position (pCMM-1) or C-terminal position (pCMM-2), GFP tagged with NanoLuc in N-terminal position (pCMM-4) or C-terminal position (pCMM-5) or GFP without any tag (pCMM-3) in addition to the paromomycin resistance gene.
We verified our construction as before, but using Bsa-I instead of Bbs-I





Lipids metabolism modification

The first step was to insert the coding sequence of the enzymes in level 0 plasmids. We ordered synthetic DNA corresponding to the coding of FAT-A, FAT-B2, DGAT and LPAAT of Elaeis guineensis (African oil palm) already verified by sequencing, but unfortunately on the four sequences we’ve order, we received only two, the sequences coding for DGAT and for LPAAT. The other two, coding for FAT-A and FAT-B2, were too rich in cytosine and guanine to be synthesized.
To speed up the process, we chose to try to build level 1 containing the enzyme straight with the synthetic DNA we received, skipping the level 0 step. After the first digestion of the plasmids we received containing DGAT or LPAAT with Bbs-I, we add the reaction mix to the reaction with others level 0 parts and started the digestion reaction with Bsa-I to make level 1 constructs. In parallel we transformed bacteria with the DNA we received and then made level 0 for both enzymes. Although we did not follow the classical way of the Modular Cloning, we still obtained clones for bacteria transformed with plasmids enabling the expressing of DGAT tagged with HiBiT in N-terminal or C-terminal proving the power of MoClo assembly. As the reactions to make level 1 plasmids with LPAAT did not work we had to do it again but in the proper way, starting with the LPAAT coding sequence in a level 0 plasmid. We made the digestion ligation reaction with Bsa-I using level 0 plasmids containing the different parts of the level 1 plasmids we wanted to build.
Again, we confirmed the presence of our insert by digesting with Bbs-I and by separating the digested DNA by electrophoresis on an agarose gel.
Thus, we obtained plasmids enabling the expression of DGAT tagged with HiBiT in N-terminal (pCM1-7) or in C-terminal (pCM1-8), or LPAAT tagged as well with HiBiT in N-terminal (pCM1-9) or C-terminal (pCM1-10).





Following the same path than before, we inserted transcriptional units with our enzymes into level M construct. This time, we used a Hygromycin resistance gene to select cells. As well, we made level M constructs with both enzymes and the Hygromycin resistance gene.
We made the digestion/ligation reaction using Bsb-I with level 1 plasmids obtained before and with level 1 plasmids enabling the expression of the hygromycin resistance gene.
We obtained plasmids enabling the expression of DGAT tagged with HiBiT in N-terminal (pCMM-6) or in C-terminal (pCMM-7), or LPAAT tagged as well with HiBiT in N-terminal (pCMM-8) or C-terminal (pCMM-9), or both DGAT and LPAAT tagged with HiBiT in N-terminal (pCMM-14) or C-terminal (pCMM-15), and for all plasmids the hygromycin resistance gene.

Part Characterization

HiBiT tag

Our goal here was to characterize the expression of the HiBiT tag in C. Reinhardtii, and show that it could be a reliable method to detect and quantify the expression of protein in this organism.

Transformation of C. Reinhardtii

Before adding the DNA to the cells, we had to digest first the plasmids containing the insert of interest to enable the insertion in the alga genome of our transcriptional units. We digested plasmids with Bsa-I and purified the DNA. The insert and the backbone were too close in size to be separated by electrophoresis on agarose gel and we did not have access to relevant restriction enzymes that would cut only in the backbone. Thus, we chose to transform cells without separating the two fragments.





Transformation was done by electroporation, and after one day of recovery, cells were cultured on media with paromomycin, selecting cells expressing the resistance gene. We observed several colonies, indicating that some algae express at least the paromomycin resistance gene.





As the DNA can be cut during the transformation and/or inserted in non-expressed region of the genome, we had to check as well the presence of the tagged GFP in cells. To do so, we screened colonies of cells using the HiBiT tag, culturing cells onto media with Nitrate as nitrogen source to activate the pNIT promoter.
Thereby, we identified colonies expressing the construct. After adding the LgBiT and the substrate we measured the bioluminescence produced by colonies, identifying colonies expressing the tagged protein, as well as pointing out colonies highly expressing the protein.





Characterization of the HiBiT tag

Now being sure that the HiBiT tag was expressed in cells, we further investigated the characteristic of the quantification by the HiBiT system. As well, we tested the activation or repression of the pNIT promoter depending on the nitrogen source.

Validation of the pNIT promoter

To be sure that the regulation of the expression of the tagged GFP was efficient, we cultured cells in media with nitrate or ammonium as nitrogen source to respectively activate or repress the expression of the tagged protein. For most of our clones, we observed, as expected, expression of the tagged protein only in media with nitrate. Interestingly, we observed a clone for which the expression was constitutive, not depending on the nitrogen source. Our hypothesis was that, in this clone, the insert was inserted in region under the control of a constitutive promoter, abolishing the regulatory properties of the promoter in the insert. Cells transformed with pCMM-4 and pCMM-5, thus expected to express a GFP tagged with NanoLuc showed very low level of expression.





Linearity of the signal

To test whether the bioluminescence signal was linear in a wide range, we cultured cells from a unique clone selected after the screening, either in media with nitrate or ammonium and diluted the cells to have a wide range of expression level. After addition of the LgBiT and substrate, we observed a linear increase of the NanoLuc activity with the number of cells.





DGAT and LPAAT

Using again the HiBiT tag, our goal was to determine whether the enzymes were expressed or not.

Transformation of C. Reinhardtii

We first digested the level M construct with Bsa-I and, after purifying the DNA, we let the DNA migrate by electrophoresis on an agarose gel. For the insert with two genes (the enzyme and the resistance gene) we could not distinguish the band corresponding to the insert and the one corresponding to the backbone so we transformed cells without separating the two fragments. For constructs with 3 genes (both enzymes and the resistance gene) we decided to stay in the same condition for the transformation and thus did not separate the DNA from insert and backbone as we have done before.
Transformation was done by electrophoresis, and after one day of recovery cells were cultured in media Hygromycin to select cells expressing the resistance gene. Some colonies grew demonstrating that at least the hygromycin resistance gene is expressed.





Cells were then cultured in media with nitrate as nitrogen source to activate the pNIT promoter. The bioluminescence activity of each colony, after addition of LgBit and substrate, was measured highlighting colonies expressing the enzymes. For cells transformed with constructs having two enzymes, we could only measure the addition of both signals without being able to distinguish the contribution of each enzymes and thus, we could not be sure that both enzymes were expressed.





Expression of the enzymes

Again, we tested the expression of the enzymes depending on the nitrogen source. For two clones, the enzymes were expressed independently of the nitrogen source.





Modification of lipid metabolism

We showed here the modifications of the lipid profile in presence of the enzymes, DGAT and LPAT.

Cell culture condition

Cells were first cultured in a medium with nitrate to activate the pNIT promoter and then, after multiple wash, they were cultured in medium without any source of nitrogen, in order to trigger the accumulation of triglycerides by cells, as described in the literature 4.
After 6 days of culture in nitrogen-free media, most of the cells died. Thus, we decided to continue to work on cells cultured in media with ammonium to try to investigate the effect of DGAT and LPAAT on the lipid profile in normal condition. As mentioned before, after the transformation, we obtained clones expressing the enzymes independently of the nitrogen source, so we used these clones, one expressing the DGAT, and the other expressing both LPAAT and DGAT, to explore the potential modification of the lipid profile.

Expression of the enzymes

To be sure that the enzymes were well expressed, we tested the presence of the enzymes thanks to the HiBiT system. We confirmed that the first clone was expressing the DGAT, and that the other clone was expressing at least one of the two enzymes, DGAT or LPAAT.





Modification of lipid profile

We investigated the modification of the lipid profile by doing a Thin Layer Chromatography (TLC) using identified lipids as standards to be compared with the profile of our clones. For the clone expressing the DGAT, we observed that one spot corresponding to the diglyceride was missing in comparison with a wild type strain making the lipid profile for this clone similar to the palm oil profile. This result is coherent with the mechanism of the DGAT. The presence of the enzyme results in an increased consumption of diglyceride, shifting the balance of lipids in the cells.





For the clone potentially expressing both DGAT and LPAAT, there was no difference in the lipid profile with wild type strain. We know, thanks to the HiBiT quantification, that this clone expresses at least one enzyme, and as well, the lipid profile of this clone was different from the profile of the DGAT-expressing clone. Thus, we thought about several hypotheses :

  • This clone does not express the DGAT, or at such a low level that it does not have any effect on the lipid profile. And so, it is expressing the LPAAT, which is coherent with the profile we observed. Indeed, if the LPAAT is expressed, we were expecting an increase of the diglyceride quantity, that we cannot detect with the TLC as it is not a quantitative method.
  • This clone expresses both DGAT and LPAAT, meaning that the decrease of the diglyceride induced by the exogenous DGAT is offset by the increase of diglyceride production induced by the exogenous LPAAT, resulting in a lipid profile similar to a wild type profile.




  1. Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. doi:10.1371/journal.pone.0016765
  2. Crozet, Pierre, et al. "Birth of a photosynthetic chassis: A MoClo toolkit enabling synthetic biology in the microalga Chlamydomonas reinhardtii." ACS synthetic biology 7.9 (2018): 2074-2086.
  3. Engler C, Kandzia R, Marillonnet S (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3(11): e3647. doi:10.1371/journal.pone.0003647.
  4. Deng, Xiaodong, Xiaowen Fei, and Yajun Li. "The effects of nutritional restriction on neutral lipid accumulation in Chlamydomonas and Chlorella." African Journal of Microbiology Research 5, no. 3 (2011): 260-270.