Measurement

Measurement is a central aspect of any synthetic biology project. One of the objectives of our project was to prove that our plasmid design could integrate and be expressed stably in the yeast Yarrowia lipolytica genome. For this, we have characterized the GFP and the RedStar2 expression levels driven by a series of pTef1 promoters in two strains of Yarrowia lipolytica. RedStar2 is particularly suitable for yeast but very little used by the scientific community compared to GFP. For GFP, calibration protocols are available which allow converting data to “universal” units of measurement. To the best of our knowledge, this was not the case for the red fluorescent proteins when we started this project. We have identified two fluorescent compounds, resorufin and rhodamine B, and successfully used them in a calibration protocol.

GFP is a fluorescent protein that emits a green color. λexcitation 488 nm andλemission 510 nm)
https://www.thermofisher.com/fr/fr/home/life-science/cell-analysis/fluorophores/green-fluorescent-protein.html
The GFP is a known reporter in the scientific community. In order to calibrate, our device for GFP absorbance measurement we had to used fluorescein molecule as a reference.
https://pubchem.ncbi.nlm.nih.gov/compound/Fluorescein.

RedStar2 is a derivative of the Red Fluorescent Protein from Discosoma sp. with improved features in terms of intrinsic brightness and speed of maturation [1]. The protein is tetrameric and contains 15 mutations compared to wild-type (Uniprot Q9U6Y8) : deltaR2, K5E, N6D, K9T, R17K, H41T, N42Q, V44A, V96I, F124L, A145P, M182K, P186Q, T202I, T217A.
This part is derived from coding sequence of RedStar2 used by Larroude et al. [2]
Redstar2 is a fluorescent protein emitting bright red light (λ of excitation at 545 nm and λ of emission at 585 nm) [1]. It is a well optimized protein for yeast [2]. It’s properties make it the ideal fluorescent protein for yeast This is why we aimed to characterize this protein in Yarrowia Lipolytica yeast.

Since Redstar2 is used very little, we lacked accurate data to establish a calibration of our machine. We have therefore chosen to use as a calibrator two proteins, the resorufin (https://pubchem.ncbi.nlm.nih.gov/compound/Resorufin#section=Crystal-Structures) (λ excitation 568 nm and 581 nm emission) [3] and rhodamine B (λ excitation 542 nm and 564 nm emission) (https://www.sigmaaldrich.com/catalog/product/sigma/83689?lang=en&region=FR) [4] emitting in wavelengths close to redstar2.

To carry out these calibration curves, we followed the 2019 igem fluorescence calibration protocol (link to the protocol pdf).The fluorescence values were normalised by OD600nm and the results are presented in figures 2. The data and error bars are the mean and standard deviation of at least three measurements (three biological replicates) figure 1 : picture representing the fluorescence protocol used to obtain the calibrations

Figure 2: particle,fluorescein, resorufin and Rhodamine B standard curves

At the end of our handling, we obtained lines indicating that the calibration was successful. Moreover, our data were verified and approved by the iGEM verification team.
One of the main concerns related to the calibration of red star2 was related to the fact that resorufin and rhodamine B also absorb at 600 nm. In order to accurately estimate the fluorescence of the compounds produced by the cells, we determine the fluorescence measurement. specific. This measurement involves both intrinsic absorption of the compound and cell growth (OD600nm)
To clarify any doubts about a potential bias on the absorbance measurement, we measured the absorbance of these compounds at 600nm. If this bias was significant, this bias could have distorted the fluorescence measurement at 600nm. and therefore the estimate of cell growth. Fortunately, we have established an average bias of 0.010 on the measure of resorufin and 0.002 on the measurement of Rhodamine B. Thus, such a bias is perfectly negligible.
Also, during absorbance measurements of resorufin and rhodamine B we observe a saturation of our measuring device for concentration values ​​higher than 2.5 μM. Indeed we have set our device to detect low concentration values ​​and have a more accurate measurement of fluorescence.
At the end of our handling, we obtained lines indicating that the calibration was successful. Moreover, our data were verified and approved by the Igem verification team.
One of the main concerns related to the calibration of red star2 was related to the fact that resorufin and rhodamine B also absorb at 600 nm. In order to accurately estimate the fluorescence of the compounds produced by the cells, we determine the fluorescence measurement. specific. This measurement involves both intrinsic absorption of the compound and cell growth (OD600nm)
To clarify any doubts about a potential bias on the absorbance measurement, we measured the absorbance of these compounds at 600nm. If this bias was significant, this bias could have distorted the fluorescence measurement at 600nm. and therefore the estimate of cell growth. Fortunately, we have established an average bias of 0.010 on the measure of resorufin and 0.002 on the measurement of Rhodamine B. Thus, such a bias is perfectly negligible.
Also, during absorbance measurements of resorufin and rhodamine B we observe a saturation of our measuring device for concentration values ​​higher than 2.5 μM. Indeed we have set our device to detect low concentration values ​​and have a more accurate measurement of fluorescence.

Measurement

We were successful at calibrating our measurement device by applying the iGEM fluorescent protocol on fluorescein and by adapting it for the calibration of resorufin and rhodamine B.Thus we were able to bring to iGEM new standardization methods that are now usefull especially in the dynamic of diversification of chassis which need different fluorescent proteins.

Improve

Inspiring and improving the work of iGEM teams from previous years is a central aspect of the iGEM competition.
At the beginning of our project, the objective was to produce in a significant quantity CLnA, we were interested in the constitutive and strong promoter. Such a promoter could promote the expression of FADX and therefore the production of punicic acid in Yarrowia lipolytica.
We started our research by the part registry of the previous years and we quickly found in the databases a promoter Part: BBa_K2117000 perfectly adapted to our expectations. This promoter of the gene TEF-1 is issued from Yarrowia lippolytica. Generally the genes TEF-1 are present in many cells and are under the control of a constitutive promoter and strongly regulating the expression of the translation elongation factor - (aplha). This protein is one of the products expressed by the cells (between 3-10% of the soluble cells)[5].
Continuing our research, we have discovered two other versions of the TEF-1 promoter:

• We found on Addgene a promoter with a sequence shorter of 6 nucleotides.
• We found on genebank a version of Ptef1 with a shorter sequence than the original sequence and an addition of 4 CACA sequence upstream to the initiator ATG.

These promoters nevertheless had a major disadvantage, the presence of a Bsa1 site. This site Bsa1 present is incompatible with the Type IIS RFC [1000] Loop assembly system that we wanted applied. Thus we mutated Bsa site 1 (GGTCTC to GGTCTg) in each promoter and formed three new promoters adapted for Type IIS RFC [1000] Loop assembly system.

One of the first questions related to the modification of promoter is to estimate if the modifications that we made to the promoters have an impact on the activity. To estimate if the promoter activity is impaired by the modifications, we used reporter genes techniques under the control of the the promoters and the reference promoters.As reporter genes we choose to use two fluorescent proteins :

• GFP (green fluorescent protein) because it is a well documented and wildly used fluorescent protein
• redstar2 This protein is also a fluorescent protein adapted for yeast but it is unfortunately far less known

Table 1. Parts used for fluorescent measurement.

Promoter’s part numbers	Expression cassettes’ part numbers	Y. lipolytica genome integration cassettes' part numbers
BBa_K2983050	BBa_K2983074	BBa_K2983181
BBa_K2983051	BBa_K2983075	BBa_K2983182
BBa_K2983052	BBa_K2983076
BBa_K2983053	BBa_K2983077
	BBa_K2983078
	BBa_K2983070
	BBa_K2983071
	BBa_K2983072
	BBa_K2983073

Experimental setup

The Y. lipolytica strains expressing Redstar2 or GFP under the control of the different promoter along with negative control (p3) and a positive control (JMY27621) were grown in either rich YPD medium or in medium glucose YNB (containing 1.7 g / L yeast nitrogen base with amino acids and ammonium sulfate, 1.5 g / L NH4Cl, 50 mM KH2PO4-Na2HPO4 buffer pH 6.8 and 60 g / L glucose). The cultivation was performed at 28 ° C in 10 ml cultivation tube 4mL of YPD medium liquid media under agitation (300 rpm) overnight.A pre-culture is made from 10μl of each strain .In fact 10 μl of each strain are disposed on transparent The cells are replaced with shaking (300rpm) at 28°C overnight. The next day 10μL of each is then transferred to COSTAR 96, an opaque 96-well polystyrene microplate. containing 90 ml of YNB liquid medium. Depending on the fluorochrome used fluorescence will be read by a Biotech (redstar2) or clariostar (GFP). In each case, the plates are incubated at 28 ° C. over three days and the fluorescence is measured at the peak of absorbance of the fluorescent compound and in parallel a second fluorescence measurement is made this time at 600 nm to estimate the growth of the cell population in each well. (OD600) Fluorescence measurements are made at regular intervals of 20 minutes.

Results

To compare the expression between each promoter, we rely on the concept of specific fluorescence. We measure the turbidity of solution at 600 nm and the fluorescence of the compound. Using this data our objective is to calculate the specific fluorescence as described in this article[6]

The difficulty in using insertion of plasmidic construction in the genome approaches is to estimate and control the insertion site and the number of copies inserted into the genome During our analyzes, we were able to group the clones according to 2 categories:

• clones presenting a copy with a fluorescence around 100 SFU / hours
• clones presenting 2 copies with a fluorescence around 200 SFU / hours

We were able to distinguish the two populations by using a reference yarrowia strain JMY22761 with one copy of the redstar2 gene.

To limit the variability on the insertion, we worked with the strain JMY2033 presenting a Zeta insertion sequence in its genome, indeed this site allows the specific recombination of the plasmic construction in the genome. It is for this reason that the strain JMY2033 have almost no clone with 2 insertions.

Once the clones were classified it was time to compare the effectiveness of the promoters. To do this we opted for bipartite student tests to compare the fluorescence averages in each strain and each plasmid. The tests are performed with a error threshold of 5%

Figure 1: Specific measurement of redstar2 in each plasmid in yarrowia lipolytica strain

Conclusion

We do not observe any significant variation of te promoter activity betwen each condition. We can deduce that [...]

Reference

[1] Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast (2004) 21, 947-962. [2] Larroude M, Park YK, Soudier P, Kubiak M, Nicaud JM, Rossignol T. A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology. Microb Biotechnol (2019) in press. [1] https://patents.google.com/patent/WO2016075312A1/en [2]: Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug;21(11):947-62. PubMed PMID: 15334558. [3] Comprehensive Analytical Chemistry, 2016 [4] https://www.sigmaaldrich.com/catalog/product/sigma/83689?lang=fr&region=FR