Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

Podsumowanie

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Streszczenie

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

Wprowadzenie

Synthetic biology aims to engineer biological systems with new functionalities useful for pharmaceutical, agricultural, and chemical industries. Assembling large numbers of DNA fragments in a high-throughput manner is a foundational technology in synthetic biology. Such a complicated process can break down into multiple levels with decreasing complexity, a concept borrowed from basic engineering sciences^1,2. In synthetic biology, DNA fragments usually assemble hierarchically based on functionality: (i) Part level: "parts" refers to DNA fragments with a specific function, such as a promoter, a coding sequence, a terminator, an origin of replication; (ii) Transcription units (TU) level: a TU consists of a promoter, a coding sequence, and a terminator capable of transcribing a single gene; (iii) Multi-gene level: a multi-gene plasmid contains multiple TUs frequently comprised of an entire metabolic pathway. This hierarchical assembly pioneered by the BioBrick community is the foundational concept for the assembly of large sets of DNAs in synthetic biology³.

In the past decade^4,5,6,7, the Golden Gate cloning technique has significantly facilitated hierarchical DNA assembly². Many other multi-part cloning methods, such as Gibson cloning⁸, ligation-independent cloning (SLIC)⁹, uracil excision-based cloning (USER)¹⁰, the ligase cycling reaction (LCR)¹¹, and in vivo recombination (DNA Assembler)^12,13, have also been developed so far. But Golden Gate cloning is an ideal DNA assembly method because it is independent of gene-specific sequences, allowing scar-less, directional, and modular assembly in a one-pot reaction. Golden Gate cloning takes advantage of type IIS restriction enzymes that recognize a non-palindromic sequence to create staggered overhangs outside of the recognition site². A ligase then joins the annealed DNA fragments to obtain a multi-part assembly. Applying this cloning strategy to the modular cloning (MoClo) system has enabled the assemble of up to 10 DNA fragments with over 90% transformants screened containing the correctly assembled construct⁴.

The MoClo system offers tremendous advantages that have accelerated the design-build-test cycle of synthetic biology. Firstly, the interchangeable parts enable combinatorial cloning to test a large space of parameters rapidly. For example, optimizing a metabolic pathway usually requires cycling through many promoters for each gene to balance the pathway flux. The MoClo system can easily handle such demanding cloning tasks. Secondly, one needs to sequence the part plasmid but typically not the TU or the multi-gene plasmids. In most cases, screening by colony PCR or restriction digestion is sufficient for verification at the TU and multi-gene plasmid level. This is because cloning the part plasmid is the only step requiring PCR, which frequently introduces mutations. Thirdly, the MoClo system is ideal for building multi-gene complex metabolic pathways. Lastly, because of the universal overhangs, the part plasmids can be reused and shared with the entire bioengineering community. Currently, MoClo kits are available for plants^{14,15,5,16,17}, fungi^{6,18,19,20,21,22}, bacteria^{7,23,24,25,26,27}, and animals^28,29. A multi-kingdom MoClo platform has also been introduced recently³⁰.

For Saccharomyces cerevisiae, Lee et al.⁶ have developed a versatile MoClo toolkit, an excellent resource for the yeast synthetic biology community. This kit comes in a convenient 96-well format and defines eight types of interchangeable DNA parts with a diverse collection of well-characterized promoters, fluorescent proteins, terminators, peptide tags, selection markers, origin of replication, and genome editing tools. This toolkit allows the assembly of up to five transcription units into a multi-gene plasmid. These features are valuable for yeast metabolic engineering, in which partial or entire pathways are over-expressed to produce targeted chemicals. Using this kit, researchers have optimized the production of geraniol, linalool³¹, penicillin³², muconic acid³³, indigo³⁴, and betalain³⁵ in yeast.

Here we provide a detailed, step-by-step protocol to guide the use of the MoClo toolkit to generate multi-gene pathways for either episomal or genomic expression. Through extensive use of this kit, we have found that the accurate measurement of DNA concentrations is key to ensuring the equimolar distribution of each part in the Golden Gate reaction. We also recommend the T4 DNA ligase over the T7 DNA ligase because the former works better with larger numbers of overhangs³⁶. Lastly, any internal recognition sites of BsmBI and BsaI must be removed or domesticated prior to assembly. Alternatively, one may consider synthesizing parts to remove multiple internal sites and to achieve simultaneous codon optimization. We demonstrate how to use this toolkit by expressing a five-gene pathway for β-carotene and lycopene production in S. cerevisiae. We further show how to knock out the ADE2 locus using the genome-editing tools from this kit. These color-based experiments were selected for easy visualization. We also demonstrate how to generate fusion proteins and to create amino acid mutations using Golden Gate cloning.

Protokół

NOTE: The hierarchical cloning protocol offered in this toolkit can be divided into three major steps: 1. Cloning part plasmids; 2. Cloning transcription units (TUs); 3. Cloning multi-gene plasmids (Figure 1). This protocol starts from the primer design and ends with applications of the cloned multi-gene plasmid.

1. Primer design for cloning the part plasmid (pYTK001):

1. Design the forward and reverse primers containing flanking nucleotides TTT at the 5' end,a BsmBI recognition site with an additional nucleotide (CGTCTCN), a 4-nucleotides (nt) overhang (TCGG) complementary to that of the entry vector, a BsaI recognition site with an additional nucleotide (GGTCTCN), and a 4-nt part-specific overhang, in addition to the template-specific sequence (Figure 2A). GoldenBraid 4.0³⁷ and GoldenMutagenesis³⁸ are some of the online software that can be used for Golden Gate specific primer design.
2. If BsaI or BsmBI recognition sites are present in any part, use a domestication step to mutate these sites prior to Golden Gate assembly³⁹. For integrative plasmids (see step 4), domesticate any NotI recognition site. To domesticate a part with one undesirable recognition site, divide the part into two subparts near the undesirable site (Figure 2A):
   1. Design the forward primer of the first subpart in the same manner as in step 1.1. but design the reverse primer with the BsmBI site and a 4-nt gene-specific overhang only.
   2. Design the second sub-part forward primer with the BsmBI site and the 4-nt gene-specific overhang only that overlaps with the reverse primer of the first sub-part. Design the reverse primer of the second sub-part in the same manner as in step 1.1.
   3. Introduce the desired mutation(s) in either the reverse (for step 1.2.1) or forward primer (step 1.2.2) at the gene-specific region of the primer.
      NOTE: Alternatively, a BsmBI- and BsaI-free and codon-optimized part can be synthesized commercially. For coding sequences (CDS), a synonymous mutation can be readily incorporated at the third nucleotide of an amino acid codon. For promoters and terminators, however, checking the mutated promoter or terminator activity using a reporter assay is recommended³⁹. If there is an undesirable restriction site toward the end of the sequence, it can be mutated using a longer reverse primer. If multiple undesirable sites are present, site-directed mutagenesis allowing mutating multiple sites may be performed⁴⁰.
3. Occasionally, fusing two proteins as a single part with a linker in between is desirable (Figure 2A). The linker helps to ensure the structural integrity of the two individual proteins⁴¹.
   1. The forward primer for the first gene is the same as in step 1.1. In the reverse primer, include a BsmBI site, a 4-nt gene-specific overhang, and a linker sequence. The 4-nt overhang can be either the linker or second gene's first few nucleotides.
   2. For the second gene, design the forward primer such that it has a BsmBI recognition site and a 4-nt overhang complementary to that of the reverse primer of the first gene. Design the reverse primer of the second gene as in step 1.1.
4. To amplify the parts by polymerase chain reaction (PCR)⁴², use a high-fidelity DNA polymerase to amplify the parts from either a genomic DNA, a cDNA, or a plasmid. Check the PCR product on a 1% agarose gel followed by gel-purification. Using purified DNA is strongly recommended, if gel purification is laborious, use at least a spin column to purify the PCR product.

2. Cloning parts into the entry vector (pYTK001) to create part plasmids (Figure 2B)

1. To set up the Golden Gate reaction mix, add 20 fmol of each PCR product and the entry vector (pYTK001), 1 μL of 10X T4 ligase buffer, 0.5 μL of Esp3I (a highly efficient isoschizomer of BsmBI), and 0.5 μL of T4 ligase. Add ddH₂O to bring the total volume to 10 μL.
2. To set up the cloning reaction, run the following program in a thermocycler: 25-35 cycles of 37 °C for 5 min (digestion) and 16 °C for 5 min (ligation), followed by a final digestion at 50 °C for 10 min and enzyme inactivation at 80 °C for 10 min.
   NOTE: 35 cycles of digestion/ligation are recommended when cloning multiple DNA pieces into the entry vector simultaneously, for example, during the cloning of fusion genes or domesticating a gene.
3. Transform the entire reaction mix into the DH5α strain or equivalent Escherichia coli chemically competent cells by heat shock. Transforming the entire 10 μL cloned product into 35 μL chemically competent E. coli cells (2 X 10⁵ cfu/mL, cfu is calculated from transforming 5 ng pYTK001 into 100 μL of the competent cells) is recommended. Spread on a lysogeny Broth (LB) plate with 35 μg/mL chloramphenicol (Cm). Incubate at 37 °C overnight.
4. After 16-18 h, take the plate out from the incubator and keep the plate at 4 °C for about 5 h to let the super folder green fluorescent protein (sfGFP) develop for a more intense green color.
5. For easier screening, place the plate on an ultraviolet (UV) or a blue light transilluminator. The sfGFP containing colonies will fluoresce under the UV light.
6. The green colonies are negative because they contain the uncut pYTK001. The white colonies are likely positive. The cloning is usually successful if there are ~30-100% white colonies. Perform further screening of a few white colonies by either colony PCR or restriction digestions (suggested enzyme: BsaI-HFv2).
7. Purify plasmids from a few of the potentially correct colonies and confirm the sequences by Sanger sequencing.

3. Assembling part plasmids into "cassette" plasmids

NOTE: A cassette plasmid contains a user-defined transcription unit (TU) consisting of a promoter, a CDS, and a terminator. A cassette plasmid allows the expression of a single gene. If the cassette plasmids will be assembled into a multi-gene plasmid, then the first step is to determine the number and the order of TUs in the multi-gene plasmid. These will determine which connectors to use in the cassette plasmids since connectors link TUs in the multi-gene plasmid. The first TU's left connector should be ConLS, and the right connector of the last TU should be ConRE. They will overlap with ConLS' and ConRE' of multi-gene plasmids. The rest of the connectors should be in the increasing numerical order. For example, if the multi-gene plasmid contains four TUs, the connector combinations would be ConLS-TU1-ConR1, ConL1-TU2-ConR2, ConL2-TU3-ConR3, and ConL3-TU4-ConRE (Figure 1).

1. Before assembling transcription units, assembling an intermediate vector with the following six parts is recommended: the left connector, the sfGFP dropout (pYTK047), the right connector, a yeast selection marker, a yeast origin of replication and the part plasmid with an mRFP1, an E. coli origin and the ampicillin-resistant gene (pYTK083) (Figure 3).
   1. Purify the above six plasmids. Record their concentrations using a UV-Vis spectrophotometer or a fluorescence-based assay and dilute each plasmid with ddH₂O so that 1 μL has 20 fmol of DNA. Calculate the DNA molar concentration by using an online calculator.
      ​NOTE: It is very important to measure the DNA concentrations accurately and to pipet precisely for the assembly to work, especially for assemblies with five to seven part plasmids. Small errors in the DNA concentration of each plasmid can cause a significant decrease in cloning efficiency.
   2. Add 1 μL of each plasmid, 1 μL of 10X T4 ligase buffer, 0.5 μL of BsaI-HFv2 (a highly efficient version of BsaI), and 0.5 μL of T4 ligase. Make up the volume to 10 μL by adding ddH₂O.
   3. To set up the cloning reaction, run the following program in the thermocycler: 25-35 cycles of 37 °C for 5 min (digestion) and 16 °C for 5 min (ligation). Omit the final digestion and heat inactivation steps as the BsaI sites need to be retained in the intermediate vector (Figure 3).
   4. Transform the entire reaction mix into the DH5α strain or other E. coli competent cells. Spread on an LB plate with 50 μg/mL carbenicillin (Cb) or ampicillin. Incubate at 37 °C overnight.
      ​NOTE: Carbenicillin is a stable analog of ampicillin.
   5. After 16-18 h, take the plate out of the incubator. The plate will contain both pale red and pale green colonies (Figures 6C and 6D). Keep the plate at 4 °C for about 5 h to let the mRFP1 and sfGFP mature. Use a UV or a blue light transilluminator to identify the green colonies, which contain the potentially correct intermediate vector.
   6. Streak out the green colonies on an LB + Cb plate and incubate at 37 °C overnight. The next day, streak out again on an LB + Cm plate and incubate at 37 °C overnight. The colonies growing on LB + Cm plates contain misassembled plasmids because Cm resistant part vectors are retained.
   7. Pick the colonies that do not grow on the LB + Cm plate and perform restriction digestions (suggested enzymes: BsaI-HFv2, Esp3I) to confirm the correctly assembled plasmid. Alternatively, use colony PCR for screening.
2. Once the intermediate vector has been successfully assembled, the next step is to assemble transcription units. This a 4-piece assembly with the following parts: the intermediate vector, a promoter, a CDS, and a terminator.
   1. Purify the four part plasmids. Record their concentrations and dilute each of plasmid so that 1 μL has 20 fmol of DNA.
   2. To set up the reaction mix, follow Step 3.1.2.
   3. To set up the cloning reaction, follow Step 2.2.
   4. Transform the entire cloning reaction mix into the DH5α or equivalent E. coli competent cells and plate on LB + Cb. Incubate at 37 °C overnight.
   5. After 16-18 h, take the plate out from the incubator. White and pale green colonies will appear (Figures 6E and 6F). Keep the plate at 4 °C for about 5 h to let the sfGFP mature. Use a UV or a blue light transilluminator to identify the non-fluorescent white colonies. These contain the potentially correct transcription units.
   6. Streak out and grow 8-10 white colonies and perform a colony PCR. Purify plasmids from the colonies that test positive from colony PCR. Carry out restriction digestion (suggested enzyme: Esp3I) to further confirm the assembly.
      ​NOTE: Sequencing transcription units is typically not necessary because the cloning involves only restriction digestion and ligation. All sequences of interest have been confirmed at the part plasmid level.

4. Assembling cassette plasmids into "multi-gene" plasmids:

NOTE: Multi-gene plasmids allow the expression of more than one gene. Depending on the downstream application, multi-gene plasmids could be replicative or integrative. Replicative plasmids have the yeast origin of replication; therefore, it can be stably maintained when yeast cell divides. Integrative plasmids do not have the yeast origin of replication. Instead, they have 5' and 3' homology arms allowing the integration of multiple genes into specific loci of the genome through homologous recombination.

1. For multi-gene plasmids, assemble an intermediate vector first.
   1. To assemble replicative intermediate vectors (Figure 4A), assemble the following six parts: the left connector (ConLS'-pYTK008), the sfGFP dropout (pYTK047), the right connector (ConRE'-pYTK072), a yeast selection marker, a yeast origin of replication, and the part plasmid with mRFP1, an E. coli origin of replication, and the kanamycin-resistant gene (pYTK084).
      1. For assembly, follow the steps from 3.1.1 to 3.1.3.
      2. Transform the entire cloning reaction mix into DH5α or equivalent E. coli competent cells, and plate on LB plus 50 μg/mL kanamycin (Km). Incubate at 37 °C overnight.
      3. For red/green color-based screening, follow step 3.1.5.
      4. For screening of misassemblies, streak and grow the green colonies on an LB + Km plate. Then follow step 3.1.6.
   2. For integrative multi-gene vectors (Figure 4B), determine the genomic locus of interest first, then design approximately 500 base pairs of 5' and 3' homology arms for integrating to that locus.
      1. Clone the 5' and 3' homology arms from yeast genomic DNA into the entry vector-pYTK001. Follow steps 1 and 2.
      2. Assemble the following seven plasmids: the left connector (ConLS'-pYTK008), the sfGFP dropout (pYTK047), the right connector (ConRE'-pYTK072), a yeast selection marker, the 3' homology arm, the part plasmid with mRFP1, E. coli origin of replication, and the kanamycin-resistant gene (pYTK090), and the 5' homology arm.
      3. For assembly and screening, follow step 4.1.1.
2. Assembly of the multi-gene plasmid
   1. Purify plasmids of the intermediate vector obtained in step 4.1 and the cassette plasmids from step 3. Record their concentrations using a UV-Vis spectrophotometer or a fluorescence-based assay. Dilute each in ddH₂O so that 1 μL has 20 fmol DNA.
   2. Add 1 μL of intermediate vector, 1 μL of each transcription unit, 1 μL of 10x T4 ligase buffer, 0.5 μL of Esp3I, and 0.5 μL of T4 ligase. Bring the volume to 10 μL of using ddH₂O.
   3. To set up the cloning reaction, follow step 2.2.
   4. Transform the entire cloning reaction mix into DH5α or equivalent E. coli competent cells, and plate on LB + Km. Incubate at 37 °C overnight.
   5. Perform the green/white screening as in step 3.2.5.
   6. Purify plasmids from a few white colonies and perform restriction digestions. Using the NotI-HF enzyme is recommended because there are two NotI sites at the E. coli origin and Km selection marker part, respectively (Figure 4B). If the assembled plasmid is very large, then another restriction site can be chosen for further confirmation. Alternatively, screen with colony PCR before proceeding to restriction digestion.

5. Applying multi-gene plasmids for chromosomal or plasmid-based gene expression

1. Integrating multi-gene plasmid into the yeast genome for chromosomal gene expression (Figure 5)
   1. Design a guide RNA (gRNA) for the desired locus. Using multiple online resources, such as Benchling, CRISPRdirect⁴³, and CHOPCHOP⁴⁴, are recommended to determine the maximum on-target specificity.
   2. Clone the synthesized 20-nt gRNA into the pCAS plasmid⁴⁵ by Gibson cloning. Linearize the pCAS plasmid by PCR using a reversed primer pair binding to the 3' of the HDV ribozyme and the 5' of the tracrRNA respectively (Figure 5).Alternatively, clone the gRNA into the sgRNA dropout plasmid (pYTK050) and assemble the dropout plasmid into a cassette plasmid with linkers. Then assemble the Cas9 TU with the Cas9 part plasmid (pYTK036). Lastly, assemble the Cas9 TU and the sgRNA TU into a replicative multi-gene plasmid.
   3. Linearize 5-15 μg of integrative multi-gene plasmid with 1 μL of NotI-HF enzyme overnight. Transform 1 μg of pCAS-gRNA and the linearized integrative multi-gene plasmid into S. cerevisiae. Prepare competent cells using either a commercially available yeast transformation kit or following the protocol by Geitz and Schiestl, 2007⁴⁶.
      ​NOTE: It is unnecessary to purify the linearized multi-gene plasmid after the NotI digestion.
   4. Pellet the cells after recovery, discard the supernatant, wash with an equal volume of water. Plate yeast cells on the complete synthetic medium (CSM) dropout plate or the yeast extract peptone dextrose medium (YPD) plate with antibiotics, depending on the yeast selective marker. Incubate at 37 °C for two days for colonies to form. In case no colony is observed, incubate for an additional one to two days at 30°C.
   5. Screen yeast colonies for integration by colony PCR⁴⁷.
   6. To cure the pCAS, streak out the colony with the correct integration onto a non-selective YPD plate. Grow at 30 °C overnight. Streak one colony from the YPD plate onto a fresh YPD plate. Again, grow at 30 °C overnight. Streak a colony from the second YPD plate to a YPD plus 100 μg/mL nourseothricin, the selection marker of pCAS. Successful curing occurs when yeast cells fail to grow on the selective plate.
      ​NOTE: If the pCAS plasmid is not cured in two rounds of non-selective YPD, streak again onto a fresh YPD for another 1-2 rounds.
2. Transforming replicative multi-gene plasmid for plasmid-based gene expression
   1. Transform 100 ng-1 μg pure multi-gene plasmid into S. cerevisiae competent cells.
   2. Plate yeast cells immediately after transformation onto the CSM dropout plate or YPD plus antibiotic plate, depending on the yeast selection marker used. Incubate at 30°C for 2-3 days for colonies to form.

Wyniki

Here the results of four replicative multi-gene plasmids for β-carotene (yellow) and lycopene (red) production. One integrative multi-gene plasmid for disrupting the ADE2 locus was constructed, the colonies of which are red.

Cloning CDSs into the entry vector (pYTK001)
ERG20 was amplified from the yeast genome and the three carotenoid genes crtE, crtYB, crtI

Dyskusje

The MoClo based cloning kit developed by Lee et al. provides an excellent resource for quick assembly of one to five transcription units into a multi-gene plasmid either for replication or integration into the yeast genome. The use of this kit eliminates the time-consuming cloning bottleneck that frequently exists for expressing multiple genes in yeast.

We tested five different conditions for the digestion/ligation cycles of Golden Gate cloning with T4 DNA ligase. We found that 30 cycles of di...

Materiały

Name	Company	Catalog Number	Comments
0.5 mm Glass beads	RPI research products	9831	For lysing yeast cells
Bacto Agar	BD & Company	214010	Component of the yeast complete synthetic medium (CSM)
Bacto Peptone	BD& Company	211677	Component of the yeast extract peptone dextrose medium (YPD)
BsaI-HFv2	New England Biolabs	R3733S	a highly efficient version of BsaI restriction enzyme
Carbenicillin	Fisher Bioreagents	4800-94-6	Antibiotic for screening at the transcription unit level
Chloramphenicol	Fisher Bioreagents	56-75-7	Antibiotic for screening at the entry vector level
CSM-His	Sunrise Sciences	1006-010	Amino acid supplement of the yeast complete synthetic medium (CSM)
Dextrose	Fisher Chemical	D16-500	Carbon source of the yeast complete synthetic medium (CSM)
Difco Yeast Nitrogen Base w/o Amino Acids	BD & Company	291940	Nitrogen source of the yeast complete synthetic medium (CSM)
dNTP mix	Promega	U1515	dNTPs for PCR
Esp3I	New England Biolabs	R0734S	a highly efficient isoschizomer of BsmBI
Frozen-EZ Yeast Trasformation II Kit	Zymo Research	T2001	For yeast transformation
Hexanes	Fisher Chemical	H302-1	For carotenoid extraction from yeast cells
Kanamycin	Fisher Bioreagents	25389-94-0	Antibiotic for screening at the multigene plasmid level
LB Agar, Miller	Fisher Bioreagents	BP1425-2	Lysogenic agar medium for E. coli culturing
LB Broth, Miller	Fisher Bioreagents	BP1426-2	Lysogenic liquid medium for E. coli culturing
Lycopene	Cayman chemicals	NC1142173	For lycopene quantification
MoClo YTK	Addgene	1000000061	Depositing Lab: John Deuber
Monarch Plasmid Miniprep Kit	New England Biolabs	T1010L	For plasmid purification from E.coli
Nanodrop Spectrophotometer	Thermo Scientific	ND2000c	For measuring accurate DNA concentrations
NotI-HF	New England Biolabs	R3189S	Restriction enzyme for integrative multigene plasmid linearization
Nourseothricin Sulphate	Goldbio	N-500-100	Antibiotic Selection marker for the pCAS plasmid used in this study
Phusion HF reaction Buffer (5X)	New England Biolabs	B0518S	Buffer for PCR using Phusion polymerase
Phusion High Fidelity DNA Polymerase	New England Biolabs	M0530S	High fidelity polymerase for all the PCR reactions
pLM494	Addgene	100539	Plasmid used to amplify crtI, crtYB and crtE used in this study
Quartz Cuvette	Thermo Electron	10050801	For quantifing carotenoids
T4 ligase	New England Biolabs	M0202S	Ligase for Golden Gate cloning
Thermocycler	BIO-RAD	1851148	For performing all the PCR and cloning reactions
Tissue Homogenizer	Bullet Blender	Model: BBX24	For homogenization of yeast cells
UV-Vis Spectrophotometer	Thermo Scientific	Genesys 150	For quantifing carotenoids
Yeast Extract	Fisher Bioreagents	BP1422-500	Component of the yeast extract peptone dextrose medium (YPD)
β-carotene	Alfa Aesar	AAH6010603	For β-carotene quantification