Supplementary Material

Transcription

1 Supplementary Material Contents 1. Tutorial on intelligent lab tube and RFIDs This tutorial explains the direct implementation of IntelliEppi, including matrix printing with sticky labels and reading these using a mobile phone with appropriate app, as well as the specialist mode using efficient lasering on the lid, upscaling, and connecting to the data logger device. The company information explains how to use professional RFIDs, if the RFID variant is chosen. This comes closer to the "internet of things". A compromise (cheaper, fully available already now) is that active RFIDs are used only for the racks with tubes as they can be larger (and more expensive). These larger RFIDs are completely satisfactory in detection (ten meter range) while the passive RFIDs are easily recorded using standard devices from shops controlling their RFID tagged goods for instance. 2. Tutorial on IntelliEppi Software This tutorial guides through the available executable for the IntelliEppi database and code. Furthermore, it provides all installation information. We also mention how easy the information from the mobile phone device or the professional data logger is recorded to your system. how easy this is switched to direct RFID input for the database; how the pseudocode has to be developed further so that the IntelliEppi software can read it. 3. Pseudocode Examples We start with simple reactions but illustrate also quite complex reactions monitored and controlled by the IntelliEppi approach.

2 TUTORIAL 1 The following brief tutorial describes one example of a simple application routine for the IntelliEppi system. A standard ml reagent tube has been chosen for the storage of isolated plasmid pet20b. The plasmid in the tube will be stored in a SMARTrack on a dedicated shelf inside a -20 C freezer in the lab. This SMARTrack no. 56 is used for the storage of various vectors. The scientist who freshly isolated pet20b at 17:10:38 on is intending to store the tube with the plasmid in row D and column 7 of SMARTrack no. 56. For rapid identification of the tube for a transformation of competent cells in the future, the tube is tagged with an on-printed data matrix code on its lid. For this purpose, IntelliEppi s data matrix-generating module ( Generator ) is used to generate a barcode encrypting ST1234SR56D7 09:54: , with 1234 being a generic number used for numerical identification of the tube. This tube information is furthermore automatically stored in the IntelliEppi data base following a simple click on the data storage button in the right-hand corner of the module. The code is then printed on solvent- and temperature-resistant label tape and fixed on the tube s lid (see S. Figure 1). Other 2D barcode formats, e.g. QR code, are also possible (but not further investigated here). The tube is then put in its corresponding spot inside SMARTrack no. 56. Figure S1: SMARTtube tagging using data matrix- or QR-code labels (last to the right). In a commercialized application, the test tube can be bought pre-labelled. Here, a data matrix is written onto the lid using Nd:YAG laser technology for instance (neodymium-doped yttrium aluminium garnet; Nd:Y3Al5O12). In this case, each tube has a unique number as identifier only and can be manually registered with the relevant information in the IntelliEppi data base (i.e. pet20b in SMARTrack 1234, stored in position D7 in SMARTrack no. 56, generated at 09:54:07 on ). In either case, the data matrix can be easily read from a convenient distance within a second or less using a smart phone app or a handy matrix reader device like Indentec s i-port H310 ISO/IEC HANDHELD INTERROGATOR. In a more commercialized version, this reader can be connected to a normal data logger device with the option of immediate manual update of relevant data connected to the tube (for instance, if the reader is connected to the IntelliEppi software on a table via Bluetooth or USB). Later in time, when the tube harbouring the isolated vector is needed, an RFID reader like Identec s i-port H310 ISO/IEC HANDHELD INTERROGATOR can be used to scan the freezer s content for SMARTrack no. 56. An active RFID tag of Identec s i-q350l FLSensorSMART-type has an in-built LED that lightens up upon scanning and therewith enables a fast and easy

3 identification of the correct SMARTrack (harbouring the searched-for tube 1234 ). Tube 1234 in position D7 (as indicated in the data base) can then be easily identified (and once again individually scanned to confirm the right identity if preferred so). The entire workflow is visually summarized in Figure S2. Figure S2: Summary for one example of a simple application routine for the IntelliEppi system. The company information provided in the supplementary material lists commercially available RFIDs suitable for individual test tube tagging that would enable an internet of things -like application. Unit prices of listed tags are yet too high for commercial scale but low enough for small scale private applications. Whereas the typically larger, active RFIDs are used for tagging of an entire rack (a SMARTrack; e.g. using Identec s i-q350l FLSensorSMART; detection can be performed from several meter distance), enlisted passive RFIDs are easily recorded using standard devices (e.g. Identec s Handheld Interrogator ), as it is commonly performed for RFID-tagged products in stores.

4 TUTORIAL 2 IntelliEppi Software Description: IntelliEppi software is user friendly and easy to install and use. It mainly consists of three modules: Generator, Tracker and Data Center (Figure S3). Figure S3: Components of IntelliEppi software application. 1. Generator produces Data Matrix barcode, having following metadata: Experiment, Researcher, Smart Tube Number, Smart Rack Number, Position, Strain and Data Time. It allows the user to generate a data matrix, print the produced data matrix and save metadata in a relational database. 2. Tracker allows the user to track tubes by loading data matrix into the IntelliEppi. Once the user loads a data matrix, the system automatically looks for related details of the tube at one or multiple positions. 3. Data Center provides complete view of metadata available in the relational database. It allows the user to search and delete data from the repository.

5 The current, released prototype version of IntelliEppi software is capable of not only reading and extracting pseudocode from its own produced data matrixes but it can also read data matrixes generated by third party devices (Figure S4). In the publically released version, there is no direct connection established to any external device for input but it can be very easily established using provided APIs. However, the user can use any market available device to track or generate data matrices. Here, IntelliEppi software can indirectly read from data matrix images. The reason behind not mentioning or using any particular external and third party device is to avoid any publicity of commercial equipment. Figure S4. Example DataMatrix. IntelliEppi Software Design and Development: IntelliEppi software is designed following principles of Butterfly model (proposed by the authors ZA and TD; It is developed using Microsoft Visual Studio, in C-Sharp programming language at Microsoft Windows 10 Platform. The database is designed and implemented in MySQL. IntelliEppi Software Installation and Execution: IntelliEppi software is freely available ( The released version of IntelliEppi can only run at the Microsoft Windows Platforms (preferably at Windows 7 and 10 etc.). It does require database connection to perform SQL based data retrieval and manipulation but can generate Data Matrix without any database support as well. To manage and track metadata about Smart tubes, it requires to either install or connect to already configured and running MySQL relational database server with IntelliEppi schema. IntelliEppi is a very simple to install and use software application. To install, it is required to download and execute setup file and follow the six-step installation process (S. Figure 5). During installation process, installer will automatically configure all required libraries and source code settings. Additionally, it requires MySQL server to be downloaded and successfully installed ( including earlier mentioned (S. Figure 3) simple one table schema (DataMatrix). To successfully test provided prototype version, following database settings are need to be followed: Server = "localhost"

6 DatabaseName = "intellieppi" UserID = "root" UserPassword = "zeeshan" User can have its own database configurations but then to successfully execute IntelliEppi, source code needs to be updated and recompiled. Figure S5. Installation steps of IntelliEppi software application. At successful installation, the user is required to click on the installed application s icon (IntelliEppi) at the desktop or execute application following a sequence of steps: Start All Programs IntelliEppi IntelliEppi.

7 Pseudo-code Examples to design reaction paths Engineering your chemical reaction path To show the full power of the software, we give here conceptual codes for different chemical reactions which show how powerful our IntelliEppi system is: The combination of software, matrix code or RFID guiding and intelligent tubes allows to achieve an equivalent of NCR guided technology for biochemical reactions: Example 1: T4 poly nucleotide kinase reaction: Position 1: Add template DNA and nucleotide mix then move to Position 2: Add T4 polynucleotide kinase, then start reaction Position 3: Count time, 30 minutes, then stop reaction Position 4: Add labelled reaction to hybridization platform at position 4 then stop reaction after 24 hours Position 5: Take filter out Position 6: Put filter in detector for read-out Example 2: Genetic Engineering of a BLUF domain fusion construct A second example shows a more complex reaction involving molecular biology genetic engineering. Fusion construct: PCR generated fragment with a light-gated BLUF domain is attached to a T4 polynucleotide kinase. This makes the T4 polynucleotide kinase light-dependent and so light can be used as an additional external control in the system. The final, generated construct is stored in Position end. Of course, the same pseudocode would for instance also generate other light-gated polymerases or exonucleases for light-gated read-in or exonuclease mediated read-out of nucleotides (which currently are expressed and tested in our laboratory) This more complex program consists of the following subroutines: Cloning of BLUF domain (1. (2. (3. (4. (5. (6. Template DNA extraction from E. coli Add suitable PCR primers; PCR the BLUF domain Easy cloning step to get BLUF domain in vector of choice Template T4 DNA, PCR the T4 polynucleotide kinase Easy cloning step to get T4 kinase domain in vector of choice Transform the vector into E. coli Expression and purification of the BLUF domain (7. Express the fusion construct (8. Purify the fusion construct on a nickel column Monitor the Activity of the new construct Position 1: Add nucleotide mix Individual steps: Add buffer, add nucleotides, add construct Position 2: Activate construct by light Here feedback monitoring of the light by light registering diode or other photosensor device is possible or

8 Position 3: Let reaction proceed for 1 minute Also, Timer function is available and integrated in the reaction flow. Position 4: Take out reaction result Then this can of course go into further work flows such as Sequence reaction product (which we routinely do to monitor construct success) A final example shows a complex synthesis process, such as dendrimer synthesis, a DNA macrame or an RNA designer aptamer transferred next into an mrna. DNA macramé For such a complex synthesis, several loops are integrated in the synthesis process: Step 1: start with basic DNA building block, Step 2: add next type of oligomer, Repeat additional option: While loop Step 3: iterative loops concatenate DNA reaction products (heat-annealing steps and/or crosslinking steps; different positions on the assembly line separate the different steps to separate temperature conditions and/or chemicals to achieve the DNA macramé) Step 4: Quality control c4.1. heck the linkage chemistry here via hybridization of DNA oligos 4.2. Automatic checking / testing of chemistry result Alternative Synthesis Route 2: Dendrimer synthesis (this would apply to different chemical dendrimers which are attached step by step. In such complex synthesis processes, the RFID monitoring could actually help to enhance accuracy of synthesis in time or positioning in space; furthermore, the tracking system allows to ensure that the chemicals are always there in time Alternative Synthesis Route 3: RNA designer aptamer and riboswitch assembly to mediate signal processing or Boolean logic switches made from RNA Starting Module 1: very similar to cloning procedure given above. Further Module 2: Branching and dendrimer synthesis as given above Route 2

9 Further Module 3: Repeated annealing and/or crosslinking steps as given above Route 1 Further Module 4: Riboswitch, cantilever or even Boolean logic designed by the different concatenated Aptamers. Step 4.1. Synthesis of the different riboswitches, cantilevers or mode complex constructs Step 4.2. IntelliEppi software modules Generator, Tracker and Data Center help now to keep track of the different fusion and modification steps so that an interesting RNA-assembly is achieved (e.g. several logical gates). Step 4.3. Quality control step (as above, again with test loops, in this case involving tests of the different aptamers) =======================================================================