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.

Transcript

This is a detailed protocol for assembling multi-gene plasmids using the Yeast MoClo kit. This method enables the convenient cloning of a variety of multi-gene classmates. It is ideal for generating a big library of related parts.

Begin by setting up the Golden Gate reaction mix. Add 20 femtomoles of each PCR product and the entry vector, one microliter of 10X T4 Ligase buffer, 0.5 microliters of Esp3I, and 0.5 microliters of T4 ligase. Add double-distilled water to bring the total volume to 10 microliters.

Then, run the cloning reaction. Transform the entire reaction mix into the DH5 alpha strain or equivalent Escherichia coli chemically competent cells by heat shock. Spread the cells on an lb plate with 35 micrograms per milliliter chloramphenicol.

Then incubate the plate at 37 degrees Celsius overnight. After 16 to 18 hours, take the plate out of the incubator and leave it at four degrees Celsius for about five hours to let the superfolder green fluorescent protein or sfGFP develop for a more intense green color. To screen the plate, place it on an ultra-violet or blue light transilluminator.

The sfGFP containing colonies will fluoresce under the UV light. The green colonies are negative because they contain the uncut part plasmid. The white colonies are likely positive.

The cloning is successful if there are 30 to 100%white colonies. Assemble an intermediate vector with the left connector, the sfGFP dropout, 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. Perform the cloning reaction as described in the text manuscript.

Transform the entire reaction mix into the DH5 alpha strain then spread the cells on an lb plate with 50 micrograms per milliliter carbenicillin or ampicillin. Incubate at 37 degrees Celsius overnight. After 16 to 18 hours, take the plate out of the incubator.

The plate will contain both pale red and pale green colonies. Keep it at four degrees Celsius for about five hours to let the mRFP1 and sfGFP mature. Use a UV or blue light transilluminator to identify the green colonies which contain the potentially correct intermediate vector.

Streak out the green colonies on an lb carbenicillin plate and incubate at 37 degrees Celsius overnight. On the next day, streak them out again on a chloramphenicol plate and incubate at 37 degrees Celsius overnight. The colonies growing on chloramphenicol plates contain misassembled plasmids.

Once the intermediate vector has been successfully assembled, proceed with assembling the transcription units. This four-piece assembly contains the intermediate vector, a promoter, a CDS, and determinator. Purify the plasmids, record their concentrations, and dilute each plasmid to 20 femtomoles of DNA per microliter.

After performing the cloning reaction, transform the entire cloning reaction mix into the DH5 alpha or equivalent E.coli competent cells and plate them on LBN carbenicillin. Then incubate the plate at 37 degrees Celsius overnight. After 16 to 18 hours, take the plate out of the incubator and keep it at four degrees Celsius for about five hours to let the sfGFP mature.

Use a UV or a blue light transilluminator to identify the non-fluorescent white colonies which contain the potentially correct transcription units. Assemble an intermediate vector for the multi-gene plasmids as described in the text manuscript. Then transform the entire cloning reaction mix into DH5 alpha cells and plate them on lb with 50 micrograms per milliliter kanamycin.

Incubate the plate at 37 degrees Celsius overnight. Perform red and green color-based screening, then streak and grow the green colonies on an lb kanamycin plate to screen for misassemblies as previously demonstrated. Next, assemble the multi-gene plasmid by setting up a cloning reaction as described in the text manuscript.

Transform the entire cloning reaction mix into DH5 alpha cells and plate them on lb kanamycin. Incubate the plate at 37 degrees Celsius overnight. Then perform the green and white screening as previously described.

This protocol was used to construct one integrative multi-gene plasmid for disrupting the ADE2 locus. Four replicative and one integrative multi-gene plasmids were assembled. The ratio of potentially correct colonies for the intermediate vector cloning was 1.83%Once the intermediate plasmid was cloned, the success rate of assembling multi-gene plasmids from the intermediate was 93.77%The negligible numbers of positive white colonies demonstrate a suboptimal assembly of multi-gene plasmids.

After transforming into yeast, colonies producing beta-carotene and lycopene grew on day three. Four colonies from each plate were streaked out onto fresh plates and grown for two more days. The carotenoids were extracted and quantified by UV-Vis spectrophotometry.

BTS1/ERG20 leads to 35 fold higher production of beta-carotene compared to the strain with ERG20 alone. Likewise, the production of lycopene is approximately 16.5 fold higher in the strain with BTS1/ERG20 compared to ERG20 alone. The multi-gene integrative plasmid was used for the disruption of the ADE2 locus, either with a gRNA and no helper DNA or with gRNA and a multi-gene integrative plasmid as helper DNA.

After three to four days, red colonies were observed on the YPD plate with Nourseothricin, indicating that ADE2 had been successfully disrupted. While attempting this method, the most important thing to remember is to measure the DNA concentrations accurately and pipette carefully while setting up this reaction mix. This technique allows researchers in the field of Yeast Metabolic Engineering to survey a large number of genes and promoters for maximized product yield.

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Journal (Bioengineering)

Journal (Bioengineering)

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

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