Introduction

1- Biological background

HiBiT Tag developed by Promega to allow for a quick method of protein quantification by luminescence (https://www.promega.com/resources/pubhub/features/hibit-a-tiny-tag-for-antibody-free-endogenous-protein-detection/).
This part is standardized in the Phytobrick MoClo standard for Chlamydomonas reinhardtii.
This tag is flanked on both side by specific fusion sites and BbsI sites to allow its integration into a level 0 plasmid of the C. reinhardtii MoClo Kit.
This tag is designed to be integrated at the B2 position (N-terminal tag).

2- Usage in iGEM projects

Bio[oil]gical Factory (iGEM Sorbonne Université 2019)

Characterization

Building the 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), allow 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.

We aimed to produce plasmids that would be used to transform C. Reinhardtii in order to characterize the N-terminal (BBa_K2909000) and C-terminal (BBa_K2909001) HiBiT tag in this organism, as well as adding a new part to the MoClo kit for this alga.

To construct plasmids expressing the N-terminal (BBa_K2909000) or C-terminal (BBa_K2909001) 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 plasmid 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 built 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 a 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 expressing transformed with our DNA, we needed to add to our construction 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 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, for the characterization of BBa_K2909000) or C-terminal position (pCMM-2, for the characterization of BBa_K2909001), 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

Figure 1: Left: Predicted agarose gel (SnapGene), pAGM8031 correspond to the acceptor vector (negative control) Right: Electrophoresis on agarose gel for two clone per construct. Ladder: Promega 200bp step ladder.

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 electrophoresis, 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.

Now being sure that the HiBiT tag was expressed in cells, we further investigate 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 increased of the NanoLuc activity with the number of cells with the construction tagged with C-terminal HiBiT (BBa_K2909001). The results were the same with the N-terminal tag (BBa_K2909000) (Data not shown)

To conclude, we have created two HiBiT tags:
BBa_K2909000: N-terminal HiBiT tag for the B2 position in the C. reinhardtii MoClo kit
BBa_K2909001: C-terminal HiBiT tag for the B5 position in the C. reinhardtii MoClo kit
That are expressed in C.reinhardtii and allows a quantifiable luminescence signal that is proportional to the quantity of tagged protein expressed in the transformed cell.
Therefore, they are working parts that allows a quick and precise measurement of protein expression.

References

Weber, E., Engler, C., Gruetzner, R., Werner, S. & Marillonnet, S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6 (2011).
Crozet, P. et al. Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synth. Biol. 7, 2074–2086 (2018).
Engler C., Kandzia R., Marillonnet S. A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLoS ONE 3 (2008).

Part:BBa_K2909000