Standardized Modular Assembly of Polycistronic Operons with Modular Cloning (MoClo) using the In-Cloning toolkit

I developed methods that allow for modular DNA assembly from small individual molecular parts up to chromosome size constructs, and to reprogram cells with these artificial chromosomes. One important innovation in the field is the development of modular cloning toolkits that allow the modular assembly of multi-gene constructs from a library of standardized molecular parts. Modular cloning toolkits are typically designed to clone monocistronic transcription units.

This protocol demonstrates a generalizable way for the assembly of polycistronic transcription units with the in-cloning toolkit. This strategy of cloning a polycistronic transcription unit allows for coding sequences to flexibly be used in either an individual transcription unit or in polycistronic arrangements. To begin, select the Level 0 parts to be assembled into a transcription unit.

These must include at least an RBS, a coding sequence, and a terminator. Choose the position in which the transcription unit will be assembled in operon. Based on the chosen position, select the corresponding acceptor plasmid and simulate the Golden Gate assembly in SnapGene.

In the Golden Gate reaction, use the enzyme SAP1 to release the ribosome binding site, coding sequence, and terminator from their respective plasmids and cut open the acceptor plasmid. Assemble the ribosome binding site, coding sequence, and terminator into the cut acceptor plasmid. Next for the Golden Gate reaction, use a pipette to add 50 femtomoles of each desired insert and 50 femtomoles of the acceptor plasmid.

One microliter each of 10x T4 DNA ligase buffer, T4 ligase, and SAP1 to a reaction tube. Then add nuclease-free water to bring the final volume to 10 microliters. Place the tube in a thermocycler.

Run 25 cycles of 37 degrees Celsius for three minutes, 16 degrees Celsius for four minutes, and 50 degrees Celsius for 20 minutes. Then heat inactivate the enzymes at 80 degrees Celsius for 20 minutes. Optionally, pipette 0.5 microliters of SAP1 to the GG reaction, and incubate at 37 degrees Celsius for one hour.

Pipette five microliters of the reaction mix into 50 microliters of chemically competent E.coli cells. Perform a heat shock transformation by plunging the tube into a water bath at 42 degrees Celsius for 30 seconds, and then place the tube on ice for two minutes. Next, incubate the cells in one milliliter of media for one hour to help recovery.

Plate 100 microliters of the transformation outgrowth onto an LB auger plate containing 100 micrograms per milliliter ampicillin. Incubate the plate overnight at 37 degrees Celsius. The next day, pick two white colonies using an inoculation loop or a pipette tip.

Inoculate each into five milliliters of LB medium containing 100 micrograms per milliliter of ampicillin. Incubate overnight at 37 degrees Celsius in a shaking incubator at 250 revolutions per minute. Perform a plaid extraction according to the manufacturer's instructions.

Digest one microgram of extracted plasma DNA by adding one microliter of 10x digestion buffer and one microliter of BBS1. Add nuclease-free water to make the total volume 10 microliters and incubate. Now load the digested plasmid on a 1%agarose gel and run electrophoresis at 100 volts for 35 minutes.

Then image the gel and confirm the correct assembly of the plasmid. To assemble a Level M construct, choose the Level 1 transcription units to be assembled. Simulate the Golden Gate assembly by releasing the first and second transcription units and the end linker using the enzyme BBS1.

Simultaneously, cut the acceptor plasmid open, then assemble all three parts into the acceptor plasmid to form the polycistronic transcription unit. The negative control for Level 1 plasmid assembly, which lacked a terminator part, yielded no colonies, whereas the complete reaction produced 292 white colonies and no red colonies. Assembly of the multi-gene construct without the second cassette at 37 degrees Celsius yielded only two white colonies while the complete assembly yielded nine large colonies and 435 small colonies.

At 30 degrees Celsius, the negative control for multi-gene assembly yielded four white colonies, while the complete reaction yielded more than 1, 500 colonies. BSA1 digest of the acceptor plasmid PMA60 gives the expected single band at 5, 500 base pairs. BSA1 digestion of plasmids from 24 colonies revealed that 23 out of 24 showed the correct restriction pattern of two bands at approximately 4, 300 base pairs and 1, 700 base pairs.

Fluorescence imaging showed strong green and cyan fluorescence in colonies from plates containing the OC6 inducer, whereas plates without an inducer showed minimal or no fluorescence.