InSillyClo

InSillyClo is made of 2 modules:

Assembly Simulator

This module simulates your Golden Gate reactions. You upload the file where you specified your genetic designs onto Assembly Simulator, along with input sequences and an auxiliary file. The algorythm simulates the cloning reaction and brings you to the result page where you can download your campaign data.

Registered users can access it at anytime in the Simulations page which contains your simulation history. For anonymous users, the results are available for the time of your web browser session.

Assembly Designer

This module allows you to create typed assemblies, which is of particular interest if you are using a MoClo system. By completing the form of Assembly Designer, you create a new assembly. You can then download the template corresponding to this assembly. This is the file where you specify your genetic designs.

Registered users can access it at anytime in the Assemblies page which contains your history, along with public assemblies.

Note that you can also use InSillyClo as a command line tool, in which the commands Template and Simulate mirror Assembly Designer and Assembly Simulator modules (documentation here).

Let's start with this simple example: the assembly of 4 different promoters with Venus reporter and tADH1 terminator.

Initialisation

• Prepare IP_mapping.csv, which maps the identifier (eg, pYT009) and the part (eg, pTDH3) of a plasmid.
• Prepare your DB of sequences, an archive containing genbank sequences. Filename must correspond to the pID column of IP_mapping.csv.
• Download from the Web App Template.xlsx from this public assembly.

You can reuse these files at each of your cloning campaigns. If needed, you will just need to update IP_mapping.csv and the DB of sequences.

Cloning Campaign

• Specify your genetic designs in the template to obtain Campaign.xlsx, using the part names present in IP_mapping.csv.
• Upload Campaign.xlsx, DB of sequences and IP_mapping.csv on the Assembly Simulator module.
• Download your campaign data from the results page.

Plasmid map

The tool computes automatically the assembly of the input sequences and generate the output sequence in genbank format.

DB produced

This file contains the standard names and types of output plasmids. It can be used for reporting and the update of iP_mapping.csv.

Dilutions

InSillyClo offers 2 methods to perform dilutions of input plasmids: direct dilution and intermediate dilution. They both allow you to obtain equimolar mix of input plasmids. All the computations are based on the following formula:

Input volume = (Final volume * Final molar concentration) / Input molar concentration

We are looking for the input volume for each input plasmid so that they are all added in the same molar quantity.

Direct dilution method

It is the simple application of the formula above.

The tool needs to know the input plasmid concentrations. You can enter mass (ng/µL) or molar (fmol/µL) concentrations directly in iP_mapping.csv (adding a column mass concentration or mol concentration). Alternatively, you can fill in the file given by the tool in the result page (Dilution section) and re-upload it once completed.

Here are the other reaction parameters you need to specify:

• Default mass concentrations of the input plasmids in the assembly (used only if you do not provide concentrations)
• The final volume of the reaction (µL)
• The final concentration of an input plasmid in the reaction (fmol/µL)
• The volume of enzyme buffer (µL)

Additionally, InSillyClo asks you the minimal tip volume. In case one input plasmid is very concentrated, the volume to add to the reaction will be very low (eg 0.12 µL). By setting minimal tip volume to higher value (eg 0.4 µL), the tool will change 0.12 to this value and apply the conversion factor to all the input plasmids to keep equimolarity. Be aware that it will cause final concentration of each input plasmid in the assembly to be higher. Alternatively, if you prefer breaking equimolarity for the highly concentrated plasmid only, you can let the minimal tip volume to 0 µL and adapt volume manually.

Intermediate dilution method

Two-step method to make dilutions easier.

You will first dilute each input plasmid to the same molar concentration (ie intermediate dilution). Then you add a fixed volume of these intermediate dilutions into the final reaction mix. Separating the dilution step from the final assembly mix is useful when input plasmid are very concentrated and when you perform many cloning in parallel.

In addition to the parameters mentioned above, you need to specify:

• the tip volume of the intermediate dilution (in µL)
• the minimal remaining volume of the intermediate dilution (in µL)

The tip volume of the intermediate dilution is the fixed volume transferred from the intermediate dilution to the final mix. At Inbio, our final volume is 10 µL and we set the tip volume of the intermediate dilution to 1 µL. Therefor the intermediate dilution is 10x concentrated, but you can adapt it to your need. The minimal remaining volume is here to ensure you to have enough volume in the intermediary dilution.

Verification

The tool simulates agarose gels after PCR or restriction digestion of the output plasmids. The DNA ladder used is GeneRuler 1kb DNA Ladder.

Simulate a PCR by uploading a file containing names and sequences of primers, then specifying which primer pair(s) to use. The PCR will also run on input plasmids, and if successful will be represented in light grey on the gel.

Users can also obtain the restriction profile by entering enzymes to be sed for the digestion.

SBOL

A SBOL file (xml format) is generated to track the assembly.

MoClo Typing System

MoClo establishes standardization of the assembly by giving a type to each part of the assembly. For instance in the Yeast Tool Kit (YTK), genes are of type 3, and overhangs are designs to ensure correct assembly.

Using a MoClo kit without its associated typing system has limitations. We show it using a cloning campaign to generate a combinatorial library of genetic designs with its auxiliary modules.

Limitations of the raw assembly solved with typed assemblies

MoClo kits often contains plasmids which have the same part (eg, Venus) with different types (3, 3a and 3b) so that you could fuse Venus to other genes. To avoid confusion, you then need to append the name of the part with its type (eg, Venus_3). This is not convenient and pollute the assembly name.

Rather than appending name with type, we suggest setting a proper type for each part of the assembly. We also include the possibility to set a character used as separator between sub-parts. For instance, the dot "." in SPsuc2.EGII indicates that SPsuc2 and EGII are 2 different sub-parts. You can choose which character to be a separator.

To add this concept of type and sub-part, you need to adapt the iP_mapping.csv and Campaign.xlsx.

• Add a "Type" column in iP_mapping.csv
• Generate a new Template.xlsx with the Assembly Designer module. Information about types and sub-parts will present in lines below the name of the input parts.

Our gitlab contains the datasets associated with the Raw and Typed assemblies.

Generate your own typed assemblies

Assembly Designer enables users to define assemblies matching characteristics of their Moclo system by choosing:

• The Assembly name
• The type IIS restriction enzyme
• The naming convention for output name
• The number and characteristics of input parts

Input part characteristics contain:

• Presence in the output name (in the YTK example, connectors are not needed in the output name since the information is already present in the type)
• Mandatory or optional (in the YTK example, some extensions allows to bypass connectors)
• Typed (the Web App associated number as default type, they can be modified in the Template.xlsx once downloaded)
• Separable (and if yes the separator character and the number of subpart)

Tailored assemblies enhance user experience with clearer, more homogeneous files. We provide public assemblies for popular MoClo systems that you can download and use directly.