This work was funded by the Research Foundation for the State University of New York (Award #: 71272) and the IMPACT Award of University at Buffalo (Award #: 000077).

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):
<ol>
	<li>Design the forward and reverse primers containing flanking nucleotides TTT at the 5&#39; 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.</li>
	<li>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):
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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&#39;s first few nucleotides.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
2. Cloning parts into the entry vector (pYTK001) to create part plasmids (Figure 2B)
<ol>
	<li>To set up the Golden Gate reaction mix, add 20 fmol of each PCR product and the entry vector (pYTK001), 1 &#956;L of 10X T4 ligase buffer, 0.5 &#956;L of Esp3I (a highly efficient isoschizomer of BsmBI), and 0.5 &#956;L of T4 ligase. Add ddH2O to bring the total volume to 10 &#956;L.</li>
	<li>To set up the cloning reaction, run the following program in a thermocycler: 25-35 cycles of 37 &#176;C for 5 min (digestion) and 16 &#176;C for 5 min (ligation), followed by a final digestion at 50 &#176;C for 10 min and enzyme inactivation at 80 &#176;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.</li>
	<li>Transform the entire reaction mix into the DH5&#945; strain or equivalent Escherichia coli chemically competent cells by heat shock. Transforming the entire 10 &#956;L cloned product into 35 &#956;L chemically competent E. coli cells (2 X 105 cfu/mL, cfu is calculated from transforming 5 ng pYTK001 into 100 &#956;L of the competent cells) is recommended. Spread on a lysogeny Broth (LB) plate with 35 &#956;g/mL chloramphenicol (Cm). Incubate at 37 &#176;C overnight.</li>
	<li>After 16-18 h, take the plate out from the incubator and keep the plate at 4 &#176;C for about 5 h to let the super folder green fluorescent protein (sfGFP) develop for a more intense green color.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Purify plasmids from a few of the potentially correct colonies and confirm the sequences by Sanger sequencing.</li>
</ol>
3. Assembling part plasmids into &#34;cassette&#34; 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&#39;s left connector should be ConLS, and the right connector of the last TU should be ConRE. They will overlap with ConLS&#39; and ConRE&#39; 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).
<ol>
	<li>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&#160;mRFP1, an E. coli origin and the ampicillin-resistant gene (pYTK083) (Figure 3).
	<ol>
		<li>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 &#956;L has 20 fmol of DNA. Calculate the DNA molar concentration by using an online calculator. 
		&#8203;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.</li>
		<li>Add 1 &#956;L of each plasmid, 1 &#956;L of 10X T4 ligase buffer, 0.5 &#956;L of BsaI-HFv2 (a highly efficient version of BsaI), and 0.5 &#956;L of T4 ligase. Make up the volume to 10 &#956;L by adding ddH2O.</li>
		<li>To set up the cloning reaction, run the following program in the thermocycler: 25-35 cycles of 37 &#176;C for 5 min (digestion) and 16 &#176;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).</li>
		<li>Transform the entire reaction mix into the DH5&#945; strain or other E. coli competent cells. Spread on an LB plate with 50 &#956;g/mL carbenicillin (Cb) or ampicillin. Incubate at 37 &#176;C overnight. 
		&#8203;NOTE: Carbenicillin is a stable analog of ampicillin.</li>
		<li>After 16-18 h, take the plate out of the incubator. The plate will contain both pale red and pale green colonies (Figures&#160;6C and 6D). Keep the plate at 4 &#176;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.</li>
		<li>Streak out the green colonies on an LB + Cb plate and incubate at 37 &#176;C overnight. The next day, streak out again on an LB + Cm plate and incubate at 37 &#176;C overnight. The colonies growing on LB + Cm plates contain misassembled plasmids because Cm resistant part vectors are retained.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>Purify the four part plasmids. Record their concentrations and dilute each of plasmid so that 1 &#956;L has 20 fmol of DNA.</li>
		<li>To set up the reaction mix, follow Step 3.1.2.</li>
		<li>To set up the cloning reaction, follow Step 2.2.</li>
		<li>Transform the entire cloning reaction mix into the DH5&#945; or equivalent E. coli competent cells and plate on LB + Cb. Incubate at 37 &#176;C overnight.</li>
		<li>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 &#176;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.</li>
		<li>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. 
		&#8203;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.</li>
	</ol>
	</li>
</ol>
4. Assembling cassette plasmids into &#34;multi-gene&#34; 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&#39; and 3&#39; homology arms allowing the integration of multiple genes into specific loci of the genome through homologous recombination.
<ol>
	<li>For multi-gene plasmids, assemble an intermediate vector first.
	<ol>
		<li>To assemble replicative intermediate vectors (Figure 4A), assemble the following six parts: the left connector (ConLS&#39;-pYTK008), the sfGFP dropout (pYTK047), the right connector (ConRE&#39;-pYTK072), a yeast selection marker, a yeast origin of replication, and the part plasmid with&#160;mRFP1, an E. coli origin of replication, and the kanamycin-resistant gene (pYTK084).
		<ol>
			<li>For assembly, follow the steps from 3.1.1 to 3.1.3.</li>
			<li>Transform the entire cloning reaction mix into DH5&#945; or equivalent E. coli competent cells, and plate on LB plus 50 &#956;g/mL kanamycin (Km). Incubate at 37 &#176;C overnight.</li>
			<li>For red/green color-based screening, follow step 3.1.5.</li>
			<li>For screening of misassemblies, streak and grow the green colonies on an LB + Km plate. Then follow step 3.1.6.</li>
		</ol>
		</li>
		<li>For integrative multi-gene vectors (Figure 4B), determine the genomic locus of interest first, then design approximately 500 base pairs of 5&#39; and 3&#39; homology arms for integrating to that locus.
		<ol>
			<li>Clone the 5&#39; and 3&#39; homology arms from yeast genomic DNA into the entry vector-pYTK001. Follow steps 1 and 2.</li>
			<li>Assemble the following seven plasmids: the left connector (ConLS&#39;-pYTK008), the sfGFP dropout (pYTK047), the right connector (ConRE&#39;-pYTK072), a yeast selection marker, the 3&#39; homology arm, the part plasmid with&#160;mRFP1, E. coli origin of replication, and the kanamycin-resistant gene (pYTK090), and the 5&#39; homology arm.</li>
			<li>For assembly and screening, follow step 4.1.1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Assembly of the multi-gene plasmid
	<ol>
		<li>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 &#956;L has 20 fmol DNA.</li>
		<li>Add 1 &#956;L of intermediate vector, 1 &#956;L of each transcription unit, 1 &#956;L of 10x T4 ligase buffer, 0.5 &#956;L of Esp3I, and 0.5 &#956;L of T4 ligase. Bring the volume to 10 &#956;L of using ddH2O.</li>
		<li>To set up the cloning reaction, follow step 2.2.</li>
		<li>Transform the entire cloning reaction mix into DH5&#945; or equivalent E. coli competent cells, and plate on LB + Km. Incubate at 37 &#176;C overnight.</li>
		<li>Perform the green/white screening as in step 3.2.5.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Applying multi-gene plasmids for chromosomal or plasmid-based gene expression
<ol>
	<li>Integrating multi-gene plasmid into the yeast genome for chromosomal gene expression (Figure 5)
	<ol>
		<li>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.</li>
		<li>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&#39; of the HDV ribozyme and the 5&#39; 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.</li>
		<li>Linearize 5-15 &#956;g of integrative multi-gene plasmid with 1 &#956;L of NotI-HF enzyme overnight. Transform 1 &#956;g 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. 
		&#8203;NOTE: It is unnecessary to purify the linearized multi-gene plasmid after the NotI digestion.</li>
		<li>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 &#176;C for two days for colonies to form. In case no colony is observed, incubate for an additional one to two days at 30&#176;C.</li>
		<li>Screen yeast colonies for integration by colony PCR47.</li>
		<li>To cure the pCAS, streak out the colony with the correct integration onto a non-selective YPD plate. Grow at 30 &#176;C overnight. Streak one colony from the YPD plate onto a fresh YPD plate. Again, grow at 30 &#176;C overnight. Streak a colony from the second YPD plate to a YPD plus 100 &#956;g/mL nourseothricin, the selection marker of pCAS. Successful curing occurs when yeast cells fail to grow on the selective plate. 
		&#8203;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.</li>
	</ol>
	</li>
	<li>Transforming replicative multi-gene plasmid for plasmid-based gene expression
	<ol>
		<li>Transform 100 ng-1 &#956;g pure multi-gene plasmid into S. cerevisiae competent cells.</li>
		<li>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&#176;C for 2-3 days for colonies to form.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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...

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 sciences1,2. In synthetic biology, DNA fragments usually assemble hierarchically based on functionality: (i) Part level: &#34;parts&#34; 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 decade4,5,6,7, 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 plants14,15,5,16,17, fungi6,18,19,20,21,22, bacteria7,23,24,25,26,27, and animals28,29. 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 &#946;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mm Glass beads</td>
			<td>RPI research products</td>
			<td>9831</td>
			<td>For lysing yeast cells</td>
		</tr>
		<tr>
			<td>Bacto Agar</td>
			<td>BD &#38; Company</td>
			<td>214010</td>
			<td>Component of the yeast complete synthetic medium (CSM)</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD&#38; Company</td>
			<td>211677</td>
			<td>Component of the yeast extract peptone dextrose medium (YPD)</td>
		</tr>
		<tr>
			<td>BsaI-HFv2</td>
			<td>New England Biolabs</td>
			<td>R3733S</td>
			<td>a highly efficient version of BsaI restriction enzyme</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Fisher Bioreagents</td>
			<td>4800-94-6</td>
			<td>Antibiotic for screening at the transcription unit level</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Fisher Bioreagents</td>
			<td>56-75-7</td>
			<td>Antibiotic for screening at the entry vector level</td>
		</tr>
		<tr>
			<td>CSM-His</td>
			<td>Sunrise Sciences</td>
			<td>1006-010</td>
			<td>Amino acid supplement of the yeast complete synthetic medium (CSM)</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td>Carbon source of the yeast complete synthetic medium (CSM)</td>
		</tr>
		<tr>
			<td>Difco Yeast Nitrogen Base w/o Amino Acids</td>
			<td>BD &#38; Company</td>
			<td>291940</td>
			<td>Nitrogen source of the yeast complete synthetic medium (CSM)</td>
		</tr>
		<tr>
			<td>dNTP mix</td>
			<td>Promega</td>
			<td>U1515</td>
			<td>dNTPs for PCR</td>
		</tr>
		<tr>
			<td>Esp3I</td>
			<td>New England Biolabs</td>
			<td>R0734S</td>
			<td>a highly efficient isoschizomer of BsmBI</td>
		</tr>
		<tr>
			<td>Frozen-EZ Yeast Trasformation II Kit</td>
			<td>Zymo Research</td>
			<td>T2001</td>
			<td>For yeast transformation</td>
		</tr>
		<tr>
			<td>Hexanes</td>
			<td>Fisher Chemical</td>
			<td>H302-1</td>
			<td>For carotenoid extraction from yeast cells</td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Bioreagents</td>
			<td>25389-94-0</td>
			<td>Antibiotic for screening at the multigene plasmid level</td>
		</tr>
		<tr>
			<td>LB Agar, Miller</td>
			<td>Fisher Bioreagents</td>
			<td>BP1425-2</td>
			<td>Lysogenic agar medium for E. coli culturing</td>
		</tr>
		<tr>
			<td>LB Broth, Miller</td>
			<td>Fisher Bioreagents</td>
			<td>BP1426-2</td>
			<td>Lysogenic liquid medium for E. coli culturing</td>
		</tr>
		<tr>
			<td>Lycopene</td>
			<td>Cayman chemicals</td>
			<td>NC1142173</td>
			<td>For lycopene quantification</td>
		</tr>
		<tr>
			<td>MoClo YTK</td>
			<td>Addgene</td>
			<td>1000000061</td>
			<td>Depositing Lab: John Deuber</td>
		</tr>
		<tr>
			<td>Monarch Plasmid Miniprep Kit</td>
			<td>New England Biolabs</td>
			<td>T1010L</td>
			<td>For plasmid purification from E.coli</td>
		</tr>
		<tr>
			<td>Nanodrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND2000c</td>
			<td>For measuring accurate DNA concentrations</td>
		</tr>
		<tr>
			<td>NotI-HF</td>
			<td>New England Biolabs</td>
			<td>R3189S</td>
			<td>Restriction enzyme for integrative multigene plasmid linearization</td>
		</tr>
		<tr>
			<td>Nourseothricin Sulphate</td>
			<td>Goldbio</td>
			<td>N-500-100</td>
			<td>Antibiotic Selection marker for the pCAS plasmid used in this study</td>
		</tr>
		<tr>
			<td>Phusion HF reaction Buffer (5X)</td>
			<td>New England Biolabs</td>
			<td>B0518S</td>
			<td>Buffer for PCR using Phusion polymerase</td>
		</tr>
		<tr>
			<td>Phusion High Fidelity DNA Polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530S</td>
			<td>High fidelity polymerase for all the PCR reactions</td>
		</tr>
		<tr>
			<td>pLM494</td>
			<td>Addgene</td>
			<td>100539</td>
			<td>Plasmid used to amplify crtI, crtYB and crtE used in this study</td>
		</tr>
		<tr>
			<td>Quartz Cuvette</td>
			<td>Thermo Electron</td>
			<td>10050801</td>
			<td>For quantifing carotenoids</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td>Ligase for Golden Gate cloning</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>BIO-RAD</td>
			<td>1851148</td>
			<td>For performing all the PCR and cloning reactions</td>
		</tr>
		<tr>
			<td>Tissue Homogenizer</td>
			<td>Bullet Blender</td>
			<td>Model: BBX24</td>
			<td>For homogenization of yeast cells</td>
		</tr>
		<tr>
			<td>UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>Genesys 150</td>
			<td>For quantifing carotenoids</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Fisher Bioreagents</td>
			<td>BP1422-500</td>
			<td>Component of the yeast extract peptone dextrose medium (YPD)</td>
		</tr>
		<tr>
			<td>&#946;-carotene</td>
			<td>Alfa Aesar</td>
			<td>AAH6010603</td>
			<td>For &#946;-carotene quantification</td>
		</tr>
	</tbody>
</table>

rapid assembly of multi-gene constructs using modular golden gate cloning

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.

Here the results of four replicative multi-gene plasmids for &#946;-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</em...

Watch this Scientific Journal Video about Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning at JoVE.com

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

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.

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

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Research

JoVE Journal

Bioengineering

12.9K Views.  State University of New York. 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.

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

ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.

A method to obtain nanofibers and complex nanostructures from single or multiple extracellular matrix proteins is described. This method uses protein-surface interactions to create free-standing protein-based materials with tunable composition and architecture for use in a variety of tissue engineering and biotechnology applications.

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...