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<p>Similar observations were obtained when using hrGFP as a reporter instead of RedStar2. These results are presented in detail on the <a href="http://parts.igem.org/Part:BBa_K2117001">BBa_K2117001</a> and <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a> parts’ Main Pages on the iGEM Registry.</p> | <p>Similar observations were obtained when using hrGFP as a reporter instead of RedStar2. These results are presented in detail on the <a href="http://parts.igem.org/Part:BBa_K2117001">BBa_K2117001</a> and <a href="http://parts.igem.org/Part:BBa_K2117005">BBa_K2117005</a> parts’ Main Pages on the iGEM Registry.</p> | ||
− | <center> <img class="img-fluid" src="https://static.igem.org/mediawiki/2019/e/e0/T--Evry_Paris-Saclay--pTef1_RedStar2_SFU.png | + | <center> <img class="img-fluid" src="https://static.igem.org/mediawiki/2019/e/e0/T--Evry_Paris-Saclay--pTef1_RedStar2_SFU.png": auto;/> </center> |
<div class="font-weight-light"><center>Figure 2. <i>In vivo</i> characterisation of RedStar2 expression driven by different pTef1 promoter variants in two <i>Y. lipolytica</i> strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates).<br></center></div> | <div class="font-weight-light"><center>Figure 2. <i>In vivo</i> characterisation of RedStar2 expression driven by different pTef1 promoter variants in two <i>Y. lipolytica</i> strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates).<br></center></div> | ||
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− | <center> <img class="img-fluid" src="https://static.igem.org/mediawiki/2019/4/45/T--Evry_Paris-Saclay--pTef1_RedStar2_Resurufin.png | + | <center> <img class="img-fluid" src="https://static.igem.org/mediawiki/2019/4/45/T--Evry_Paris-Saclay--pTef1_RedStar2_Resurufin.png": auto;/> </center> |
<div class="font-weight-light"><center>Figure 3. <i>In vivo</i> characterisation of RedStar2 expression driven by different pTef1 promoter variants in two <i>Y. lipolytica</i> strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates).<br></center></div> | <div class="font-weight-light"><center>Figure 3. <i>In vivo</i> characterisation of RedStar2 expression driven by different pTef1 promoter variants in two <i>Y. lipolytica</i> strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates).<br></center></div> | ||
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− | <center> <img class="img-fluid" src="https://static.igem.org/mediawiki/2019/2/28/T--Evry_Paris-Saclay--pTef1_RedStar2_RhB.png | + | <center> <img class="img-fluid" src="https://static.igem.org/mediawiki/2019/2/28/T--Evry_Paris-Saclay--pTef1_RedStar2_RhB.png": auto;/> </center> |
<div class="font-weight-light"><center>Figure 4. <i>In vivo</i> characterisation of RedStar2 expression driven by different pTef1 promoter variants in two <i>Y. lipolytica</i> strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates).</p> | <div class="font-weight-light"><center>Figure 4. <i>In vivo</i> characterisation of RedStar2 expression driven by different pTef1 promoter variants in two <i>Y. lipolytica</i> strains. The data and error bars are the mean and standard deviation of at least 6 measurements (at least three biological replicates each measured as two technical duplicates).</p> |
Revision as of 15:49, 21 October 2019
![](https://static.igem.org/mediawiki/2019/c/c1/T--Evry_Paris-Saclay--BannierePromotersFluoProteins.png
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Improving promoters
Getting inspired by and improving the work of iGEM teams from previous years is a central aspect of the iGEM competition.
The objective of our project is to produce medically-relevant Conjugated Linolenic Acids (CLnAs), which is a class of rare fatty acids with three conjugated bonds. In this project, we developed a launchpad for their bioproduction using the oleaginous yeast Yarrowia lipolytica, a powerful chassis organism, whose metabolism is naturally poised for lipid production. CLnAs are synthetized by bifunctional fatty acid conjugase / desaturase (FadX) enzymes from linoleic acid, a natural metabolite for our chassis. Thus, to convert it into a CLnA, only the presence of a FadX enzyme is necessary.
To drive the expression of FadX in Yarrowia lipolytica, the choice of the promoter is an important step: constitutive or inducible ? weak, medium or strong ? Several natural and synthetic promoters were characterized for Yarrowia lipolytica that allow tunable heterologous gene expression in this chassis (for a review see [1]). As producing a CLnA may lead to perturbations of vital cellular lipids (like the membrane glycerophospholipids) and thus reduce cell viability, we decided not to use an extremely strong promoter (like hp4d for instance [2]), nor a medium one (to put all odds on our side).
We started our research from iGEM’s part registry and we quickly found in the database the pTef1 promoter (BBa_K2117000), a constitutive promoter native for the oleaginous yeast Y. lipolytica. It is a strong promoter that controls the expression of the translation elongation factor-1 alpha [3], a protein that is one of the most expressed in most cells (between 3-10% of the soluble proteins [4]). We will refer to BBa_K2117000 as pTef1a.
This promoter seemed suitable for our project, but had a major disadvantage: the presence of a BsaI site that makes it incompatible with the Type IIS RFC[1000]-compatible Loop assembly system that we designed for Yarrowia lipolytica. To circumvent this incompatibility with the RFC[1000] standard, we mutated the BsaI site (GGTCTC) to GGTCTg and thus created a new compatible part, BBa_K2983050, that we’ll refer to as pTef1c.
A quick sequence analysis of BBa_K2117000 revealed several differences compared to wild-type pTef1 promoter (nucleotides 1227374 to 1226969) of Yarrowia lipolytica W29 chromosome C (GenBank Acc. n° CP028450.1). Three of the four mutations were introduced by the iGEM16_DTU-Denmark in order to remove two illegal restriction sites for BioBrick RFC[10]-compatibility (SpeI, PstI). As these sites are accepted in the Type IIS RFC[1000] standard, we created a closer to wild-type version of pTef1 promoter, BBa_K2983051, that has also a mutated BsaI site (GGTCTC to GGTCTg) which makes this part compatible with the iGEM Type IIS RFC[1000] standard. We will refer to BBa_K2983051 as pTef1d.
Continuing our research, we discovered another version of the pTef1 promoter, which is shorter and Type IIS RFC[1000] compatible [5,6]: BBa_K2983052, that we’ll refer to as pTef1e.
Unpublished observations of our PI, Jean-Marc Nicaud, suggest that the presence of a 4 nucleotide sequence CACA just upstream the ATG of the gene may lead to increased gene expression. Thus, we added BBa_K2983053 to the list of pTef1 variants to test. We will refer to BBa_K2983053 as pTef1f.
A sequence comparison of all pTef1 variants is presented in figure 1. Apart from the above mentioned promoters, we included BBa_K2117000+scar, as this promoter was used to build BBa_K2117005 by standard 3A assembly which leaves TACTAG as a scar between the promoter and the ATG of the downstream gene. We will refer to BBa_K2117000+scar as pTef1b.
![](https://static.igem.org/mediawiki/2019/4/45/T--Evry_Paris-Saclay--pTef1_alignment.png)
One of the main questions related to the modification of a promoter sequence is related to the impact it may have on its activity. To estimate if the pTef1 promoter activity is impaired by the modifications highlighted in Figure 1, we used a fluorescent reporter gene.
For this, we decided to use RedStar2, as it is the “brightest and most yeast-optimized version of the red fluorescent protein” [7]. We placed RedStar2 under the control of a pTef1 promoter variant and of the Lip2 terminator (BBa_K2983055). The resulting transcriptional units (BBa_K2983075, BBa_K2983076, BBa_K2983077, BBa_K2983078) were assembled into our YL-pOdd1 plasmid (BBa_K2983030,) which is part of our Loop assembly system dedicated to our chassis, the oleaginous yeast Y. lipolytica (for further details on this system, visit the dedicated page on this wiki ). Thus, we generated four RedStar2 expression plasmids (BBa_K2983175, BBa_K2983176, BBa_K2983177, BBa_K2983178) able to integrate upon transformation, into a Y. lipolytica Po1d stain. To be able to make comparisons with the expression driven by the pTef1 variant already in the registry (pTef1a, BBa_K2117000), the BBa_K2983175 was subject to site directed mutagenesis to restore the BsaI site and thus generate BBa_K2983174.
Promoter labels | Promoter’s part numbers | RedStar2 expression cassettes’ part numbers | Y. lipolytica genome integration cassettes' part numbers |
---|---|---|---|
pTef1a | BBa_K2117000 | BBa_K2983074 | BBa_K2983174 |
pTef1c | BBa_K2983050 | BBa_K2983075 | BBa_K2983175 |
pTef1d | BBa_K2983051 | BBa_K2983076 | BBa_K2983176 |
pTef1e | BBa_K2983052 | BBa_K2983077 | BBa_K2983177 |
pTef1f | BBa_K2983053 | BBa_K2983078 | BBa_K2983178 |
Experimental setup
For pTef1 promoter characterization we decided to use the auxotrophic wild-type Y. lipolytica strain JMY195 [8], but also JMY2033 [9]. JMY195 is a Po1d strain, thus, by the means of the Zeta sequences [10], the Y. lipolytica genome integration cassettes (table 1) will be inserted randomly. JMY2033 is a derivative of JMY195 that contains a zeta docking platform at the ura3-302 locus. In this strain, the insertion is not random, but site specific which limits the risks of multiple insertion of plasmidic constructions in the genome.
These two Y. lipolytica strains were transformed with the NotI digested RedStar2 expression plasmids (BBa_K2983174, BBa_K2983175, BBa_K2983176, BBa_K2983177, BBa_K2983178). As a negative control, we also transformed them with the NotI digested empty YL-pOdd1 vector (BBa_K2983030, ). As positive control we used the JMY7621 stain [11] which contains a single genome copy of a RedStar2 expression cassette.
For fluorescence measurements, yeast cells were first grown overnight in rich YPD medium then diluted by 100x in YNB-glucose medium (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, 10 g/L glucose and 0.1 g/L leucine) in an opaque wall 96-well polystyrene microplate, the COSTAR 96 (Corning). The plate was incubated at 28°C at 200 rpm and the fluorescence (λexcitation 558 nm and λemission 586 nm) and OD600nm were measured every 15 min during 300 cycles in a SynergyMx (BioTek) plate reader.
To compare the expression between each promoter, we rely on specific fluorescence [11]. We measure the turbidity of the culture at 600 nm and the fluorescence of RedStar2, and determine the mean rate of fluorescence/OD600nm (SFU/h) increase during the exponential growth phase. This method allows quantifying RedStar2 expression in a manner independent of the length of the lag phase. Using the calibration curves presented on the dedicated page of this wiki, we converted the arbitrary units into Molecules of Equivalent Resorufin (MEResorufin) / particle and Molecules of Equivalent Rhodamine B (MERhB) / particle.
Results
The results of the different pTef1 promoter strength quantifications are presented in Figures 2, 3 and 4 using different units of measure.
For nearly each construct, we obtained and analysed twice at least 3 clones (and up to 32), the majority of each displaying comparable specific fluorescence values similar to the value obtained for the positive control. However, a minority of clones were constantly displaying twice more SFU/h suggesting they have integrated 2 copies of the RedStar2 expression cassette.
Indeed, the difficulty in using insertion of plasmid construction in the genomic approaches is to estimate and control the insertion site and the number of copies inserted into the genome. In the auxotrophic wild-type Y. lipolytica strain JMY195, our RedStar2 expression cassettes integrate randomly and the protein expression may vary depending on the insertion site. In the JMY2033 strain, the insertion occurs mainly at the zeta docking platform at the ura3-302 locus.
As can be easily observed from the results presented in Figures 2, 3 and 4, the different modifications of the pTef1 promoter sequence highlighted in Figure 1 do not have a drastic impact on its activity. The specific fluorescence values are similar between the different variants of pTef1 in both Y. lipolytica strains and independent of copy number / genome.
To confirm these observations, we performed a two-tailed student test with an error threshold of 5% in order to compare the specific fluorescence averages in each strain and each construct. No statistically significant difference was observed between the different constructs in both strains and at both copy number / genome.
Similar observations were obtained when using hrGFP as a reporter instead of RedStar2. These results are presented in detail on the BBa_K2117001 and BBa_K2117005 parts’ Main Pages on the iGEM Registry.
![](https://static.igem.org/mediawiki/2019/e/e0/T--Evry_Paris-Saclay--pTef1_RedStar2_SFU.png)
![](https://static.igem.org/mediawiki/2019/4/45/T--Evry_Paris-Saclay--pTef1_RedStar2_Resurufin.png)
![](https://static.igem.org/mediawiki/2019/2/28/T--Evry_Paris-Saclay--pTef1_RedStar2_RhB.png)
Conclusion
We have successfully built 4 versions of the pTef1 promoter: pTef1c (BBa_K2983050), pTef1d (BBa_K2983051), pTef1e (BBa_K2983052) and pTef1f (BBa_K2983053) which are able to drive the expression of a reporter gene in the oleaginous yeast Yarrowia lipolytica at an equivalent strength as the part already present in the iGEM Registry, pTef1a (BBa_K2117000).
Thus, we have made an improvement of the BBa_K2117000, a version of pTef1 promoter not compatible with the Type IIS RFC[1000].
References
[1] Larroude M, Rossignol T, Nicaud JM, Ledesma-Amaro R. Synthetic biology tools for engineering Yarrowia lipolytica. Biotechnol Adv (2018) 36, 2150-2164.[2]Madzak C, Tréton B, Blanchin-Roland S. Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol. 2000 Apr;2(2):207-216.
[3]Müller S, Sandal T, Kamp-Hansen P, Dalbøge H. Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast (1998) 14, 1267-1283.
[4]Celińska E, Ledesma-Amaro R, Larroude M, Rossignol T, Pauthenier C, Nicaud JM. Golden Gate Assembly system dedicated to complex pathway manipulation in Yarrowia lipolytica. Microb Biotechnol (2017) 10, 450-455.
[5]Moore SJ, Lai HE, Kelwick RJ, Chee SM, Bell DJ, Polizzi KM, Freemont PS. EcoFlex: a multifunctional MoClo kit for E. coli synthetic biology. ACS Synth Biol (2016) 5, 1059-1069.
[6]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) 16, 1249-1259.
[7]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.
[8] Barth G, Gaillardin C. Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology. Springer, Berlin (1996) 1, 314-388.
[9]Lazar Z, Rossignol T, Verbeke J, Crutz-Le Coq AM, Nicaud JM, Robak M. Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications. J Ind Microbiol Biotechnol (2013) 40, 1273-1283.
[10]Pignède G, Wang HJ, Fudalej F, Seman M, Gaillardin C, Nicaud JM. Autocloning and amplification of LIP2 in Yarrowia lipolytica. Appl Environ Microbiol (2000) 66, 3283-3289.
[11] Park YK, Korpys P, Kubiak M, Celinska E, Soudier P, Trébulle P, Larroude M, Rossignol T, Nicaud JM. Engineering the architecture of erythritol-inducible promoters for regulated and enhanced gene expression in Yarrowia lipolytica. FEMS Yeast Res (2019) 19, foy105.