Difference between revisions of "Part:BBa K2117005"

Revision as of 13:48, 21 October 2019

Device encoding TEF1 and hrGFP for expression in Yarrowia lipolytica

This is a composite part consisting of the TEF1 promoter (BBa_K2117000) and hrGFP (BBa_K2117003).

This part encodes the humanized Renilla reniformis green fluorescent protein (hrGFP) codon-optimized for Y. lipolytica and the native Y. lipolytica constitutive promoter TEF1.

Studies have shown the hrGFP to be functional in Y. lipolytica under the expression of the TEF1 promoter measured by flow cytometry (1).

Usage and Biology

The green fluorescent protein originates from the sea pansy, Renilla reniformis. This part can be used as a reporter protein for protein expression in the yeast chassis, Y. lipolytica. It is compatible with all the iGEM Biobrick standards.

• Emission: 500-600 nm

• Excitation: 488 nm

Sequence and Features

Functional Parameters

• Emission: 500-600 nm

• Excitation: 488 nm

Experimental data

The two parts (TEF1 and hrGFP) was assembled with the pSB1A8YL backbone (BBa_K2117009) using standard 3A assembly and transformed into E. coli. The construct was confirmed using restriction analysis (See Figure 1), PCR and sequencing (data not shown).

Figure 1: Analytical digestion of the pSB1A8YL containing the this TEF1-hrGFP device. The fragment lengths can be seen on the ladder, and the restriction enzyme and predicted fragment lengths is stated above the fragments.

Afterwards, the construct was transformed into Y. lipolytica PO1f and grown on plates containing selective media. Single colonies were picked and grown in liquid selective media, and subjected to fluorescence microscopy. Figure 2 shows the Y. lipolytica PO1f cells under a confocal laser microscope with 100x magnification. The high fluorescent output from this construct, proves that the cells are producing hrGFP, and ultimately that our expression system consisting of pSB1A8YL (BBa_K2117009) and the TEF1 promoter (BBa_K2117000) can be used for heterologous protein expression in Y. lipolytica.

Figure 2: Fluorescence microscopy conducted by a confocal laser microscope with 100x magnification. A and D are taken using standard brightfield, B and E are taken using the GFP filter and with the excitation laser on and C and F are overlays of the two photos where the black bagground has been removed (C is an overlay of A and B, and F is an overlay of D and E). A, B and C are Y. lipolytica PO1f cells with this GFP expressing device shuttled by our plasmid pSB1A8YL, (BBa_K2117009). D, E and F are Y. lipolytica PO1f cells with the empty pSB1A8YL plasmid, which serves as a control for the GFP signal. Notice that even though the empty vector control shows trace amounts of auto-fluoresence the strain with the GFP expressing device clearly exhibits higher levels of fluorescence, which proves that our expression system works as intended.

References: 1) Blazeck, J., Liu, L., Redden, H., Alper, H. (2011). Tuning Gene Expression in Yarrowia lipolytica by a Hybrid Promoter Approach. APPLIED AND ENVIRONMENTAL MICROBIOLOGY.

Characterization by Evry Paris-Saclay 2019

In the context of iGEM 2019 competition, we developed a metabolic engineering projet for the production of medically-relevant Conjugated Linolenic Acids (CLnAs) and used as a chassis the oleaginous yeast Yarrowia lipolytica, an organism whose metabolism is naturally poised for lipid production. For the expression of our enzymes, we choose the pTef1 promoter, a constitutive promoter native for Y. lipolytica. It is a strong promoter that controls the expression of the translation elongation factor-1 alpha [1], a protein that is one of the most expressed in most cells (between 3-10% of the soluble proteins [2]).

Our bibliographic research led us to designing and characterisation of several versions of the pTef1 promoter (Figure 1 and Table 1).

We started our research from iGEM’s part registry and we quickly found in the database the pTef1 promoter (BBa_K2117000), that we’ll refer to as pTef1a.

This promoter has been used to build this part (BBa_K2117005) by standard 3A assembly which leaves TACTAG (BBa_G0000) as a scar between the promoter and the ATG of the downstream gene. We will refer to BBa_K2117000+scar as pTef1b.

pTef1 (BBa_K2117000) 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 Y. lipolytica. To circumvent this incompatibility with the RFC[10] 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 Y. 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 [3,4]: 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.

Figure 1. Sequence comparisons of the six pTef1 promoter variants. The alignment was generated using the MUSCLE algorithm implemented in SnapGene.

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: either hrGFP (BBa_K2117001) or RedStar2 (BBa_K2983060).

It's in this context that we characterized this part and present here the results, in a manner that allows easy comparaisons with the similar other parts in the registry (listed in Table 1).

To allow expression in Y. lipolytica, we equipped this part with the Lip2 terminator (BBa_K2983055) and assembled the resulting transcriptional unit BBa_K2983070 into our YL-pOdd1 plasmid (BBa_K2983030) which is part of our Loop assembly system dedicated to Y. lipolytica. Thus we generated BBa_K2983170 able to integrate upon transformation, into an Y. lipolytica Po1d stain.

Similarly, we placed hrGFP (BBa_K2117001) under the control of pTef1c (BBa_K2983050) and of pTef1e (BBa_K2983052) and of the Lip2 terminator (BBa_K2983055). The resulting transcriptional units (BBa_K2983072 and BBa_K2983073) were assembled into our YL-pOdd1 plasmid (BBa_K2983030) which is part of our Loop assembly system dedicated to Y. lipolytica. Thus we generated BBa_K2983172 and BBa_K2983173 able to integrate upon transformation, into an Y. lipolytica Po1d strain.

To be able to make comparisons with the expression driven by the pTef1 variant already in the registry (pTef1a, BBa_K2117000), the BBa_K2983172 was subject to site directed mutagenesis to restore the BsaI site and thus generate BBa_K2983171.

All these parts are summarized in Table 1.

For this part characterization we decided to use the auxotrophic wild-type Y. lipolytica strain JMY195 [5], but also JMY2033 [6]. JMY195 is a Po1d strain, thus, by the means of the Zeta sequences [7], 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 hrGFP expression plasmids (BBa_K2983170, BBa_K2983171, BBa_K2983172 and BBa_K2983173). As a negative control, we also transformed them with the NotI digested empty YL-pOdd1 vector (BBa_K2983030). 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 483 nm and λemission 530 nm) and OD600nm were measured every 10 min during 500 cycles in a CLARIOstar (BMGLabtech) plate reader. To compare the expression between each promoter, we rely on specific fluorescence [8]. We measure the turbidity of the culture at 600 nm and the fluorescence of hrGFP (BBa_K2117001), and determine the mean rate of fluorescence/OD600nm (SFU/h) increase during the exponential growth phase. This method allows quantifying hrGFP (BBa_K2117001) expression in a manner independent of the length of the lag phase.

Using the calibration curves presented in Figures 2 and 3 (and as an excel file too), we converted the arbitrary units into Molecules of Equivalent FLuorescein (MEFL) / particle (Figure 4).

Figure 2. Particle standard curve.

Figure 3. Fluorescein standard curve.

The results of this part characterization are presented in Figure 4 together with the characterization of the other similar parts in the registry (listed in Table 1).

As can be easily observed, 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.

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.

Figure 4. In vivo characterisation of hrGFP expression driven by different pTef1 promoter variants in two Y. lipolytica 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)

Thus, we confirm that the presence of the scar TACTAG between the pTef1a promoter (BBa_K2117000) and hrGFP (BBa_K2117001) as it is in this part (BBa_K2117005) does not affect promoter activity. This observation highlights the versatility and the robustness of pTef1 and makes him stand in the first row of Y. lipolytica promoters choices.

References

[1] 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.

[2] Cavallius J, Zoll W, Chakraburtty K, Merrick WC. Characterization of yeast EF-1 alpha: non-conservation of post-translational modifications. Biochim Biophys Acta (1993) 1163, 75-80.

[3] 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.

[4] 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.

[5] Barth G, Gaillardin C. Yarrowia lipolytica. In: Wolf K (ed) Non conventional yeasts in biotechnology. Springer, Berlin (1996) 1, 314-388.

[6] 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, 1273-83.

[7] 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.

[8] 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) in press.