Biofoundry-assisted Golden Gate cloning with AssemblyTron

Golden Gate assembly is a requisite method in synthetic biology that facilitates critical conventions such as genetic part abstraction and rapid prototyping. However, compared to robotic implementation, manual Golden Gate implementation is cumbersome, error-prone, and inconsistent for complex assembly designs. AssemblyTron is an open-source python package that provides an affordable automation solution using open-source Opentrons OT-2 lab robots. Automating Golden Gate assembly with AssemblyTron can reduce failure-rate, resource consumption, and training requirements for building complex DNA constructs, as well as indexed and combinatorial libraries. Here, we dissect a panel of upgrades to AssemblyTron’s Golden Gate assembly capabilities, which include Golden Gate assembly into modular cloning part vectors, error-prone PCR combinatorial mutant library assembly, and modular cloning indexed plasmid library assembly. These upgrades enable a broad pool of users with varying levels of experience to readily implement advanced Golden Gate applications using low-cost, open-source lab robotics.


Introduction
Golden Gate assembly is a principal enabling technology in synthetic biology, and automated implementation has transformed it into a high throughput genetic engineering tool.Golden Gate assembly is a scarless DNA assembly strategy capable of assembling up to 40 fragments (1)(2)(3)(4).Plasmid libraries of standardized modular parts are often used to facilitate rapid Golden Gate assembly, circumventing PCR amplification.Originally, the Modular Cloning system (MoClo) implemented this strategy and has inspired a wide array of plasmid toolkits for different applications (5)(6)(7)(8)(9), which have been reviewed by Casini et al. (10).Modular cloning toolkits define a standard of unique and seamless overhangs for predefined part types, which are then used to build libraries of genetic parts such as promoters, terminators, markers, coding sequences, etc that can be assembled in combinatorial fashion.In the Sky biofoundry, RoboMoClo combines lab automation with the MoClo system to create a biofoundry-assisted Golden Gate platform for metabolic pathway-level DNA assembly (11).However, RoboMoClo does not provide openly available code, requires expensive robotics, and is intended as a service platform rather than a transferable tool.
Prior to AssemblyTron, resources for automating customized, in-lab Golden Gate workflows were scarce.Golden Gate assembly is often performed with commercial kits, where just one or two fragments are inserted into a predefined entry vector -a gross underutilization of the method.Customized Golden Gate greatly expands the flexibility and potential construct complexity, however manual Golden Gate implementation requires careful design and extensive molecular biology background knowledge (3).Fortunately, many automation tools exist for designing custom Golden Gate assemblies (12)(13)(14)(15), but manually interpreting and implementing their output files is labor intensive and error-prone.Modular cloning toolkits, such as the MoClo system, can simplify Golden Gate workflows, however PCR is unavoidable for integrating a gene of interest into a part plasmid.Moreover, the complex, multi-level nature of toolkits limits their accessibility for inexperienced researchers.We designed AssemblyTron to help resolve these issues with automation.
AssemblyTron is an open-source python package that, when paired with an Opentrons OT-2 lab robot, can provide biofoundry capabilities with minimal startup investment (16).AssemblyTron 1.0 uses j5 DNA design files as input and generates fully customized OpenTrons Application Program Interface (API) protocol scripts for assembling plasmids via homology mediated assembly or Golden Gate.We previously validated that AssemblyTron is adept at automating multi-part Golden Gate assembly and can simultaneously increase throughput, eliminate human errors, and reduce training demands (16).AssemblyTron adds to a very sparse field of open-source build tools for automating Golden Gate assembly.Since our initial publication, we have expanded AssemblyTron's toolkit of capabilities.
Here, we will review AssemblyTron 2.0 upgrades for automating biofoundry-enabling Golden Gate protocols.First we describe a modified version of AssemblyTron's standard Golden Gate assembly protocol, which accepts any Type IIS restriction-enzyme-digestible part entry vector as the construct backbone.Next, we introduce a protocol for assembling mutant libraries with Golden Gate.In this strategy, AssemblyTron uses error-prone PCR with tunable error-rates to mutate user-specified DNA fragments that are assembled into plasmid libraries via Golden Gate.Finally, we introduce the modular cloning toolkit builder, which is the most recent and developmental installment of AssemblyTron.This feature provides an interactive graphical user interface (GUI), inspired by Figure 2 of the Yeast MoClo Toolkit publication (7).The current toolkit builder offered by AssemblyTron is for the auxin modular cloning toolkit, which is a custom, in-house toolkit in the Wright Lab (see Note 1).However, our automation strategy for toolkit building can be adapted for any toolkit system as a fork or pull-request of the AssemblyTron github repository https://github.com/PlantSynBioLab/AssemblyTron.AssemblyTron was developed and debugged using Windows OS, so we recommend implementing it with Windows OS. (see Note 3).The most up to date version of AssemblyTron is available at https://github.com/PlantSynBioLab/AssemblyTron.Prior to installing AssemblyTron, users need the latest versions of Python and R installed on their OT-2 connected computers.The package can also be downloaded from pypi.We recommend using Anaconda to easily install and manage AssemblyTron dependencies, which are listed in section 2.2.The OpenTrons run app should also be installed for uploading AssemblyTron scripts to the OT-2.

Plasmid Design and preliminary setup
A plasmid design must be prepared with j5 and DIVA prior to building it with AssemblyTron.For entry vector backbone parts, the forced assembly strategy should be set to "digest."Eugene rules should be added to specify which parts belong together, unless a complete combinatorial design is desired.When running the j5 algorithm, the design strategy should be set as "Combinatorial Golden Gate." (see Note 4).After running j5, download results, unzip the compressed file, and store the design folder on your local hard drive.Primers can be found in masteroligolist.csvor in the pmas00001_combinatorial.csv design file and must be ordered prior to moving forward.To ensure successful PCR amplification, digest any circular template plasmids with a single-cutter restriction endonuclease prior to PCR amplification (see Note 5).Optionally, entry vectors can be digested with type IIS restriction endonuclease and gel purified to reduce template vector carry-through.This step can be skipped if the entry vector contains a toxicity gene (e.g.ccdB) or a fluorescent marker that distinguishes it from positive transformants.

Entry Vector Golden Gate Assembly
AssemblyTron enables flexible Golden Gate assembly into any entry vector, which is a critical capability for assembling level 0 part plasmids for modular toolkits as well as reducing dependency on commercial Golden Gate kits.Gate assembly setup, and Golden Gate assembly run.This option makes it easy to skip certain parts of the protocol without modifying python code.For example, primers may already be diluted, so that part of the protocol would not need to be selected.8. Select confirm.The gradient optimization algorithm runs automatically, unless the touchdown PCR option was specified.9. Screen the reaction_setup.txtfile, which specifies where to place tubes for PCRs and final assemblies.Ensure that each PCR and assembly is assigned to a tube and close the file until needed.10.The setup script imports the primer dilution and Golden Gate protocol writer modules (dilution_24_digests_writer.py and GoldenGate_digests_separatepcrruns_gradient_writer.py) and executes them automatically.The final customized protocol scripts then open automatically.Close them to allow the setup script to finish running.11.Navigate to the new directory, labeled with the current date, that was just generated.12. Protocol scripts are ready to be uploaded directly to the OT-2.(see Note 10).13.See section 3.5 for instructions on uploading protocols to the OT-2.14. Amplify fragments, run Golden Gate assembly, clean and concentrate assembled plasmids, and transform into high efficiency chemically competent E. coli.

Mutant Library Assembly
Mutant library screening is a common strategy for protein engineering and directed evolution (reviewed by Wang et al.) (19).AssemblyTron provides protocols for facilitating mutant library assembly with error-prone PCR and Golden Gate assembly.Targeted mutagenesis of gene regions followed by assembly with unaltered backbone fragments provides high-quality plasmid libraries for functional screening (20,21).Using AssemblyTron to automate library assembly ensures consistent library quality, which saves time and resources.AssemblyTron allows users to tune error rate (Figure 1A), which accommodates various target fragment sizes and libraries of varying degeneracy.We assembled libraries of pink chromoprotein expression plasmids with different error rates and screened for functional variation by colony color (Figure 1B, 1C).Our data indicates that increasing error-rate settings with AssemblyTron correlate with increasing loss-of-function colorless colonies and diminishing pink colonies.

Figure 1: Tuning mutagenesis with AssemblyTron error prone PCR. A) A dialog box provided by
AssemblyTron is used to tune the error rate.B) Qualitative mutant accumulation increases as higher mutation rates are specified with AssemblyTron.C) Quantitative mutant accumulation increases as higher mutation rates are specified based on a plot of normalized colony count for 10X dilutions of plates in B.
Here, we provide a detailed protocol for using AssemblyTron's library assembly feature.Our workflow consists of three stages.In stage one, an initial PCR is performed to amplify backbone fragments and a template amplicon for the error-prone fragment with high fidelity polymerase (see Note 11).In stage two, error-prone PCR is used to mutate the target fragment with a user-specified error rate (Figure 1A).In stage three, Golden Gate is used to assemble the mutant fragment pool with backbone fragments.
Golden Gate Primers with 5' Golden Gate overhangs (see Note 12), should be ordered prior to implementing this protocol.Additionally, shorter primers lacking 5' Golden Gate overhangs must be ordered to generate the initial template amplicon in stage one.This template will be used in stage two error-prone PCR.Primers lacking Golden Gate overhangs prevent the initial template amplicon from being assembled into the library in stage three, which would decrease the overall error-rate of the library.

Stage 1
1. Navigate to the ~/AssemblyTron/src/AssemblyTron/ directory and go to the Error_prone_PCR_Golden_Gate/ folder.2. Select a stage one setup script.1_Setup_STD_GoldenGate_digests_PCR.py is for libraries that will be assembled into entry vector backbones, and 1_Setup_STD_GoldenGate_PCR.py is for libraries that will be assembled strictly from PCR amplicons.(see Note 13).Click on the appropriate script.3. Use the dialog box to navigate to your j5 design folder, which was previously saved to the local hard drive (section 3.1).Select confirm, and AssemblyTron automatically parses the j5 design file by importing a parsing module (see Note 7). 4. Screen the reagent_setup.txtfile, which will be used to stage primers, templates, reagents, labware, and tubes on the OT-2 deck.Ensure that all templates and primers are assigned to a deck position.Close it until needed.(see Note 14).

A parameter selection dialog box with default values for PCR and assembly is displayed.
Most defaults do not need to be modified, however the following parameters will require consideration or adjustment: a. Check the Gradient PCR or touchdown PCR option.Stage one PCR is set to the gradient option by default.This is to ensure clean bands with high DNA concentration for high library quality and successful yields after gel extraction.
(see Note 15).b.Template concentrations (ng/μL) must be entered to proceed.6. Select confirm once parameters are updated.7.In the next dialog box, select which parts of the protocol to run.Users can choose from Dilution and PCR mix in this stage, since assembly will not be performed until stage three.(see Note 16).8. Screen the reaction_setup.txtfile, which specifies where to place PCR tubes on the ondeck thermocycler.Ensure that each PCR is assigned to a tube and close the file until needed.9.The setup script imports the primer dilution and Golden Gate protocol writer modules (dilution_24_digests_writer.py and GoldenGate_digests_separatepcrruns_gradient_writer.py) and executes them automatically.The final customized protocol scripts open automatically.Close them to allow the setup script to finish running.10.Navigate to the New dated folder (e.g., 202310011038_PCR/).

Navigate to the
~/AssemblyTron/src/AssemblyTron/Error_prone_PCR_Golden_Gate/ directory and go to the dated PCR folder generated in stage one (e.g., 2023010011038_PCR/). 26.Click on the stage three setup file (e.g., 3_2023010011038_Assembly.py) 27.Use the dialog box to navigate to your j5 design folder, which was previously saved to the local hard drive (section 3.1).Select confirm, and AssemblyTron automatically parses the j5 design file by importing a parsing module (see Note 7).28.Screen the reagent_setup.txtfile, which will be used to stage reagents and labware on the OT-2 deck.Close it until needed.29.A parameter selection dialog box with default values for assembly is displayed.Most defaults do not need to be modified, however the following parameters will require consideration or adjustment: a. Check the "PaqCI?"option.This specifies whether PaqCI or a different type IIS restriction enzyme is being used for Golden Gate assembly.PaqCI requires an activator additive, which alters reagent volumes in the final Golden Gate assembly mixes.Select confirm.30.In the next dialog box, select which parts of the protocol to run.Users can choose from Golden Gate setup and Golden Gate run in stage three, since PCR steps were performed in stages one and two.31.Use the next dialog box for uploading concentrations of each assembly fragment in ng/μL.Each entry box is labeled by well number to avoid confusion.A new window will appear for each assembly (e.g., if there are four assemblies, four concentration input windows will appear consecutively).32.Screen the reaction_setup.txtfile, which specifies where to place fragment PCR tubes and final assembly PCR tubes on the on-deck thermocycler.Close the file until needed for setting up the OT-2 deck.33.The setup script imports the Golden Gate protocol writer module (GoldenGate_digests_separatepcrruns_gradient_writer.py) and executes it automatically.The final customized protocol script opens automatically.Close it to allow the setup script to finish running.34.Navigate to the new stage three folder that was just created (e.g., 3_202310021038_Assembly/). 35.The final customized protocol script that is ready to be uploaded directly to the OT-2.
(see Note 10).See section 3.5 for details on uploading protocols to the OT-2.36.Run the library assembly on the OT-2, clean and concentrate, and transform into high efficiency competent E. coli cells.

Modular Cloning toolkit builder
Automated modular cloning indexed plasmid library assembly has proved to be a successful platform strategy for biofoundries (11).Combinatorial part assembly and shuffling will inevitably remain a useful cloning strategy even as DNA synthesis prices become trivial, so we have begun offering it as an AssemblyTron feature.Here we introduce a preliminary, opensource protocol for automating construct assembly from an auxin modular toolkit we have built in-house, based on part standards of the Yeast Modular Cloning Toolkit (7).This toolkit build strategy will serve as a model for expanding AssemblyTron platform to other modular cloning toolkits in the future.3. Use the dialog box to specify how many construct variants you are assembling from the toolkit.4. A graphical user interface for selecting modular parts appears (Figure 2).Choose the desired combination of parts.One part from each column must be selected, for a total of eight (or more, see Note 21) parts (Figure 2A, 2B). 5. Select confirm after selecting parts for the first construct.New selection windows will be consecutively displayed for specifying each additional construct.6.A parameter selection dialog box with default values for assembly is displayed.Most defaults do not need to be modified.Select confirm.7. Navigate to the new dated folder that was just created (e.g., 202310021254_MoClo/). 8. Run the MoClo_writer.pyprotocol writer script by clicking on it.This will generate the final customized protocol script that is ready to be uploaded directly to the OT-2.9. See section 3.5 for details on uploading protocols to the OT-2.10.Run construct assembly on the OT-2, clean and concentrate, and transform into high efficiency chemically competent E. coli.
14. Use the shortened 3' only primers lacking Golden Gate overhangs for the initial template amplicon to be used in stage 2. 15.Touchdown PCR isn't a good option for error-prone PCR stage 1 since it is more likely to amplify off-target fragments and sometimes doesn't provide the highest possible concentration of amplified product.16.There is no need for methylated templated digestion with DpnI in the error-prone PCR workflow since all fragments are gel extracted.17.Gel purification reduces template carry-through and ensures a high quality library at the conclusion of the workflow.18. Remember to use the amplicon from stage 1 as template for error-prone PCRs in stage 2. 19.There is no need for methylated templated digestion with DpnI in the error-prone PCR workflow since mutant fragments are amplified from a PCR amplicon with no Golden Gate overhangs.20.PCR tubes for error-prone amplification should be placed in the same location on the thermocycler as their initial template amplicons from stage 1. 21.A combination of a type 3a and a type 3b part may be selected in place of a standard type 3 part to make an N-terminal protein fusion.Similarly, a type 4a and a 4b part combination may be selected in place of a standard type 4 part to make a C-terminal protein fusion.22.The Opentrons run app provides a deck map under labware setup>map view.23.Ensure that shortened 3' only primers lacking Golden Gate overhangs are used to generate the initial template amplicons in error-prone PCR stage one.Backbone fragments should be amplified from standard Golden Gate overhang primers in stage one.

Figure 2 : 1 .
Figure 2: AssemblyTron graphical user interface for modular part selection.A) Any combination of eight color-coded part types can be specified for assembly.B) A pair of type 3a and 3b parts can be selected in place of a standard type 3 part for N-terminal protein fusions.Similarly, a type 4a and 4b part can be selected in place of a standard type 4 part for a C-terminal protein fusion.

1 .
Navigate to the ~/AssemblyTron/src/AssemblyTron directory on your local drive.Go to the Golden_Gate/ folder, and select the Setup_digests_gradient.py script.(seeNote 6). 2. Use the dialog box to navigate to your j5 design folder, which was previously saved to the local hard drive (section 3.1).Select confirm, and AssemblyTron automatically parses the j5 design file by importing a parsing module (see Note 7). 3. Screen the reagent_setup.txtfile,which will be used to stage primers, templates, reagents, labware, and tubes on the OT-2 deck.Ensure that all templates and primers are assigned to a deck position.Close it until needed.4.A parameter selection dialog box with default values for PCR and assembly is displayed.Most defaults do not need to be modified, however the following parameters will require consideration or adjustment: is large, it is safer to use the gradient amplification option.)b.Check the "PaqCI?"option.This specifies whether PaqCI or a different type IIS restriction enzyme is being used for Golden Gate assembly.PaqCI requires an activator additive, which alters reagent volumes in the final Golden Gate assembly mixes.c.There are two blank parameter entry windows available so that users can add additional parameters to protocols without modifying the dialog box generator scripts.These do not have to be entered.d.Entry windows for template concentrations (ng/μL) must be entered to proceed. 5. Select confirm once parameters are updated.6.A dialog box is displayed for entering concentrations of the entry vector backbones used in this assembly.Enter concentrations in ng/μL and select confirm.(see Note 9). 7.In the next dialog box, select which parts of the protocol to run.Users can choose from Primer and template dilution, PCR preparation, DpnI digest (template removal), Golden a. Check the Gradient PCR or touchdown PCR option.The default is touchdown PCR, but this may need to be changed depending on the desired DNA yield (see Note 8) or how prone the PCR is to failure (e.g., if the range of annealing temperatures for fragments