|
|
Line 1: |
Line 1: |
| {{Marburg}} | | {{Marburg}} |
| <html> | | <html> |
− | <style>
| + | <style> |
| .box-dark { | | .box-dark { |
− | background-color: #3d404d;
| + | background-color: #3d404d; |
− | min-height: 30vh;
| + | min-height: 30vh; |
− | box-shadow: 1px 1px 40px black;
| + | box-shadow: 1px 1px 40px black; |
− | margin-left: -10vw;
| + | margin-left: -10vw; |
− | width: 120vw;
| + | width: 120vw; |
− | position: relative;
| + | position: relative; |
− | z-index: 2;
| + | z-index: 2; |
− | display: flex;
| + | display: flex; |
− | flex-direction: column;
| + | flex-direction: column; |
− | align-items: center;
| + | align-items: center; |
− | transform: rotate(355deg);
| + | transform: rotate(355deg); |
− | justify-content: center;
| + | justify-content: center; |
− | margin-top: -12vh;
| + | margin-top: -12vh; |
| } | | } |
| | | |
| .heading { | | .heading { |
− | color: #f5f5f5;
| + | color: #f5f5f5; |
− | text-align: center;
| + | text-align: center; |
− | font-size: 1.75em;
| + | font-size: 1.75em; |
− | width: fit-content;
| + | width: fit-content; |
− | margin-top: 25px;
| + | margin-top: 25px; |
− | margin-bottom: unset !important;
| + | margin-bottom: unset !important; |
− | transform: rotate(-355deg);
| + | transform: rotate(-355deg); |
| } | | } |
| | | |
| .line { | | .line { |
− | border-top: 2px solid #f5f5f5;
| + | border-top: 2px solid #f5f5f5; |
− | background-color: #f5f5f5;
| + | background-color: #f5f5f5; |
− | border-width: 2px;
| + | border-width: 2px; |
− | display: block;
| + | display: block; |
− | width: 100px;
| + | width: 100px; |
− | margin-top: 25px;
| + | margin-top: 25px; |
− | margin-bottom: unset;
| + | margin-bottom: unset; |
− | transform: rotate(-355deg);
| + | transform: rotate(-355deg); |
| } | | } |
| | | |
| .logo { | | .logo { |
− | width: 100px;
| + | width: 100px; |
− | height: 100px;
| + | height: 100px; |
− | position: absolute;
| + | position: absolute; |
− | bottom: -50px;
| + | bottom: -50px; |
− | transform: rotate(-355deg);
| + | transform: rotate(-355deg); |
− | margin-left: -10px;
| + | margin-left: -10px; |
| } | | } |
| | | |
| .main { | | .main { |
− | overflow-x: hidden;
| + | overflow-x: hidden; |
| } | | } |
| | | |
| hr { | | hr { |
− | display: block;
| + | display: block; |
− | height: 1px;
| + | height: 1px; |
− | border: 0;
| + | border: 0; |
− | border-top: 2px solid #3d404d;
| + | border-top: 2px solid #3d404d; |
− | padding: 0;
| + | padding: 0; |
− | margin: 1em auto;
| + | margin: 1em auto; |
− | width: 50vw;
| + | width: 50vw; |
| } | | } |
| | | |
| @media (max-width: 810px) { | | @media (max-width: 810px) { |
| | | |
− | .logo,
| + | .logo, |
− | .line,
| + | .line, |
− | .heading {
| + | .heading { |
− | margin-left: -30px;
| + | margin-left: -30px; |
− | }
| + | } |
| | | |
− | .line {
| + | .line { |
− | margin: 1.5rem 0 !important;
| + | margin: 1.5rem 0 !important; |
− | margin-left: -40px !important;
| + | margin-left: -40px !important; |
− | }
| + | } |
| } | | } |
− | | + | </style> |
− | </style>
| + | <div> |
− | <div>
| + | |
| <div class="box-dark"> | | <div class="box-dark"> |
− | <h1 class="heading">
| + | <h1 class="heading"> |
− | L A B A U T O M A T I O N
| + | L A B A U T O M A T I O N |
− | </h1>
| + | </h1> |
− | <hr class="line">
| + | <hr class="line"> |
− | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg"
| + | <img src="https://static.igem.org/mediawiki/2019/a/ac/T--Marburg--logo.svg" class="logo" alt="Syntex Logo"> |
− | class="logo"
| + | |
− | alt="Syntex Logo">
| + | |
| </div> | | </div> |
| <div style="margin-top: 10vh;"> | | <div style="margin-top: 10vh;"> |
− | <section class="section">
| + | <section class="section"> |
− | <h1 class="title">A.P.P Automated Purfication Protocol</h1>
| + | <h1 class="title">A.P.P Automated Purfication Protocol</h1> |
− | <p style="text-align: justify;">
| + | <p style="text-align: justify;"> |
− | Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS)
| + | Vielleicht noch ein allgemeinem abstract zu Messung (vergleiche andere WIKIS) |
− | </p>
| + | </p> |
− | </section>
| + | </section> |
− | <section class="section">
| + | <section class="section"> |
− | <article>
| + | <article> |
− | <h1 class="title">Storytelling:</h1>
| + | <div> |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | This year’s iGEM Team worked extensively on automating a plasmid purification on Opentrons’ OT-2. Plasmid
| + | This year’s iGEM Team worked extensively on automating a plasmid purification on Opentrons’ |
− | purification is an indispensable part of completing the cloning workflow in the OT-2.</p>
| + | OT-2. Plasmid |
− | <figure style="float: right; margin-left: 25px;">
| + | purification is an indispensable part of completing the cloning workflow in the OT-2.</p> |
− | <img style="height: 400px; width: 600px"
| + | <figure style="float: right; margin-left: 25px;"> |
− | src="https://static.igem.org/mediawiki/2019/6/60/T--Marburg--SyntexConnections.png"
| + | <img style="height: 400px; width: 600px" |
− | alt="Connections between Opentrons, Promega and QInstruments">
| + | src="https://static.igem.org/mediawiki/2019/6/60/T--Marburg--SyntexConnections.png" |
− | <figcaption style="max-width: 600px">
| + | alt="Connections between Opentrons, Promega and QInstruments"> |
− | Fig.1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and QInstruments.
| + | <figcaption style="max-width: 600px"> |
− | </figcaption>
| + | Fig.1 - iGEM team Marburg 2019 is establishing connections between Opentrons, Promega and |
− | <br>
| + | QInstruments. |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | </figcaption> |
− | Since the time of an iGEM project is limited to only one year, consequently only a limited amount of work can be
| + | <br> |
− | done in that time, which is even reduced by failing experiments and making mistakes in the lab. To overcome this
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
− | problem and increase the reproducibility and simultaneously raise the amount of experiments in the lab, we
| + | Since the time of an iGEM project is limited to only one year, consequently only a limited |
− | automated plasmid purification on the OT-2. Using this protocol and making it open-source <b>(GitHub Link?)</b>,
| + | amount of work can be |
− | we
| + | done in that time, which is even reduced by failing experiments and making mistakes in the |
− | achieved to parallelize work in the lab or make more time for public engagement, human practice, IHP or
| + | lab. To overcome this |
− | everything else not directly lab-related, benefiting the whole iGEM community. This benefits will also be
| + | problem and increase the reproducibility and simultaneously raise the amount of experiments |
− | translated beyond iGEM community such as in the amateur biohackers, enthusiasts, and students community and even
| + | in the lab, we |
− | to research groups doing cutting-edge research.<br>
| + | automated plasmid purification on the OT-2. Using this protocol and making it open-source |
− | This idea started when we found out that there is also a great need in the industry for an automated cloning
| + | <b>(GitHub Link?)</b>, |
− | workflow. Promega provided us with great advice <b>(Link to IHP)</b> and sponsored the Wizard® MagneSil® Plasmid
| + | we |
− | Purification System, QInstruments sponsored the BioShake D30-T elm and Opentrons sponsored their Magnetic
| + | achieved to parallelize work in the lab or make more time for public engagement, human |
− | Module. Through our work aligned with the philosophy of iGEM for nurturing collaborations, we enabled
| + | practice, IHP or |
− | connections between these companies to achieve the true potential of their products. This kind of bridge would
| + | everything else not directly lab-related, benefiting the whole iGEM community. This benefits |
− | not have been possible otherwise.<br>
| + | will also be |
| + | translated beyond iGEM community such as in the amateur biohackers, enthusiasts, and |
| + | students community and even |
| + | to research groups doing cutting-edge research.<br> |
| + | This idea started when we found out that there is also a great need in the industry for an |
| + | automated cloning |
| + | workflow. Promega provided us with great advice <b>(Link to IHP)</b> and sponsored the |
| + | Wizard® MagneSil® Plasmid |
| + | Purification System, QInstruments sponsored the BioShake D30-T elm and Opentrons sponsored |
| + | their Magnetic |
| + | Module. Through our work aligned with the philosophy of iGEM for nurturing collaborations, |
| + | we enabled |
| + | connections between these companies to achieve the true potential of their products. This |
| + | kind of bridge would |
| + | not have been possible otherwise.<br> |
| + | <br> |
| + | Nevertheless, a massive amount of barriers had to be broken down. The shaker was a bit |
| + | bigger than the space |
| + | normally occupied by modules in the OT-2 and needed stabilizing support, so it was obvious |
| + | to design a |
| + | custom-made shaker adapter and print it with our own in-house 3D printer, which would keep |
| + | the costs for the |
| + | automation of this workflow extremely low. Moreover, the 3D design will be publicly |
| + | available in our GitHub |
| + | repository (LINK), which will make our solution accessible to everyone with access to a 3D |
| + | printer.<br> |
| + | <br> |
| + | Additionally, we stumbled across serious problems with the calibration of our OT-2 and |
| + | accessing the shaker with |
| + | the pipette. The BioShake D30-T elm is currently not a usual labware defined by Opentrons’, |
| + | so we had to be |
| + | creative and come up with our own labware definition. Opentron is recently rolling out a |
| + | major update from their |
| + | OT-2 3.9 to 4.0 firmware that includes a lot of paradigm changes, making it impossible for |
| + | us to define it as a |
| + | decent custom labware. That is why we came up with the idea to use Opentrons’ internal |
| + | coordinate system and |
| + | defining the required 96 Deep Well Plate on the shaker as coordinates. This facilitated |
| + | accessing the shaker |
| + | with the pipette, being as precise as Opentrons’ own labware definitions, but a whole series |
| + | of problems |
| + | followed, as we tried to use Opentrons’ pipette functions to transfer the chemicals. We |
| + | managed these problems |
| + | as well, by defining our own Python functions, telling the pipette how to transfer liquids |
| + | from and to the |
| + | defined shaker. In the end when running the script, one would not be able to tell the |
| + | difference between the |
| + | labware and functions defined by us from the ones defined by Opentrons’.<br> |
| + | <br> |
| + | </p> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and |
| + | scientists we wanted to |
| + | show the best conditions for cultivation and the best measuring method for our parts in UTEX |
| + | 2973. Therefore |
| + | we analyzed a big variety of cultivating conditions in measuring growth curves, tried to |
| + | find a standard in |
| + | light measurement, evaluated different reporters???, established a measurement method and |
| + | compared it to a |
| + | already known FACS measurement method (?). |
| + | </p> |
| + | <p style="text-align: justify; margin-bottom: 1em;"> |
| + | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 |
| + | μE. [quelle]. |
| + | So we had to measure the light conditions in our incubators and while doing this simple task |
| + | the first |
| + | part of standardization began. We discovered that nearly every paper? is using different |
| + | methods to measure |
| + | their light conditions and that it is a really complex and important procedure. So we got in |
| + | contact with |
| + | cyano and light measurement experts [link IHP] to confront this problem and standardize it. |
| + | In the following |
| + | popup we show different ways of measurement, their (dis-)advantages and different results |
| + | depending on the |
| + | measuring instrument.<br> |
| + | Not only the light intensity but also a variety of other cultivating parameters needed to be |
| + | analyzed. |
| + | In literature and while talking with different experts (IHP), we recognized that small |
| + | deviations of these |
| + | parameters had a huge impact on the growth speed of Synechococcus elongatus. While |
| + | establishing UTEX 2973 as |
| + | a new chassis we evaluated this impact on the growth speed and were able to show |
| + | combinations of parameters |
| + | that lead to the fastest growth speed.<br> |
| + | Another aspect was measuring the expression and characterize our part. Different |
| + | possibilities were |
| + | discussed and after testing them we decided on two methods in our project (plate reader and |
| + | FACs). One |
| + | approach was to measure the fluorescence/luminescence with a plate reader [link part |
| + | measurement]. Plate |
| + | readers belong to standard equipment of every lab nowadays, and could deliver easy |
| + | reproducible results.<br> |
| + | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) |
| + | [link facs]. In |
| + | contrast to a platerader a FACs device delivers results with high accuracy by measuring |
| + | every cell by its |
| + | own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side |
| + | not |
| + | every laboratory posses a FACs/device. So in the end we would like to offer a two method |
| + | analyzed database |
| + | from our crontructs for iGEM teams and research groups, who do not have access to a FACS and |
| + | show the |
| + | difference in measurement methods.<br> |
| + | At the end of the project we were able to create a protocol how to handle Synechococcus |
| + | elongatus UTEX 2973 |
| + | and make a contribution to the cyano community by establishing essential/fixed standards in |
| + | measurement. |
| + | </p> |
| + | <figure align=center> |
| + | <img style="height: 500px; width: 300px" |
| + | src="https://static.igem.org/mediawiki/2019/b/bb/T--Marburg--opentrons_magnetic_module.JPG" |
| + | alt="OT-2 left"> |
| + | <img style="height: 500px; width: 300px" |
| + | src="https://static.igem.org/mediawiki/2019/3/30/T--Marburg--opentrons_shaker.JPG" |
| + | alt="OT-2 right"> |
| + | <figcaption style="max-width: 1400px"> |
| + | Fig.2 - Single-Channel pipette, magnetic module and shaker in action while performing |
| + | the plasmid |
| + | purification. |
| + | </figcaption> |
| + | </figure> |
| + | <br> |
| + | <p> |
| + | Putting the pieces together, we were able to translate the manual plasmid purification |
| + | protocol provided by Nans |
| + | Bodet into an Opentrons protocol, being the very first of its kind. We pioneered a workflow |
| + | for up to six |
| + | samples with the p300 Single-Channel Electronic Pipette and a scaled-up version for up to 48 |
| + | samples with the |
| + | p300 8-Channel Electronic Pipette without having to intervene even once. This scalability |
| + | provides important |
| + | flexibility for various kinds of experiments.<br> |
| + | <br> |
| + | In our process of developing and running the protocol we determined some problems on |
| + | increasing the yield of our |
| + | plasmids. There was a large number of parameters that could be varied, changing the final |
| + | concentration of the |
| + | plasmids. For example, we realized that the duration of lysis is paramount for the yield and |
| + | success of plasmid |
| + | purification. Over-lysis will lead to a decrease in plasmid yield, whereas under-lysis will |
| + | induce clumping of |
| + | magnetic beads; thus failing the experiment. After a whole heap of plasmid purifications we |
| + | managed to identify |
| + | the most relevant parameters and improve the protocol in the best way possible.<br> |
| + | <br> |
| + | </p> |
| + | <figure align=center> |
| + | <img style="height: 700px; width: 600px" |
| + | src="https://static.igem.org/mediawiki/2019/e/ea/T--Marburg--SingleChannelSetup.png" |
| + | alt="OT-Layout left"> |
| + | <img style="height: 700px; width: 600px" |
| + | src="https://static.igem.org/mediawiki/2019/d/df/T--Marburg--8channelSetup.png" |
| + | alt="OT-Layout right"> |
| + | <figcaption style="max-width: 1400px"> |
| + | Fig.3 - Final setup for the automated plasmid purification workflows in the OT-2. The |
| + | left picture shows the |
| + | setup for the single channel workflow, the right picture for the 8-channel workflow. |
| + | </figcaption> |
| + | </figure> |
| + | |
| + | <video src="https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--PlasmidPurificationMarburg.mp4" |
| + | controls poster="vorschaubild.jpg"></video> |
| + | </div> |
| + | </article> |
| + | </section> |
| + | </div> |
| + | </div> |
| + | |
| <br> | | <br> |
− | Nevertheless, a massive amount of barriers had to be broken down. The shaker was a bit bigger than the space
| |
− | normally occupied by modules in the OT-2 and needed stabilizing support, so it was obvious to design a
| |
− | custom-made shaker adapter and print it with our own in-house 3D printer, which would keep the costs for the
| |
− | automation of this workflow extremely low. Moreover, the 3D design will be publicly available in our GitHub
| |
− | repository (LINK), which will make our solution accessible to everyone with access to a 3D printer.<br>
| |
| <br> | | <br> |
− | Additionally, we stumbled across serious problems with the calibration of our OT-2 and accessing the shaker with | + | </body> |
− | the pipette. The BioShake D30-T elm is currently not a usual labware defined by Opentrons’, so we had to be
| + | |
− | creative and come up with our own labware definition. Opentron is recently rolling out a major update from their
| + | |
− | OT-2 3.9 to 4.0 firmware that includes a lot of paradigm changes, making it impossible for us to define it as a
| + | |
− | decent custom labware. That is why we came up with the idea to use Opentrons’ internal coordinate system and
| + | |
− | defining the required 96 Deep Well Plate on the shaker as coordinates. This facilitated accessing the shaker
| + | |
− | with the pipette, being as precise as Opentrons’ own labware definitions, but a whole series of problems
| + | |
− | followed, as we tried to use Opentrons’ pipette functions to transfer the chemicals. We managed these problems
| + | |
− | as well, by defining our own Python functions, telling the pipette how to transfer liquids from and to the
| + | |
− | defined shaker. In the end when running the script, one would not be able to tell the difference between the
| + | |
− | labware and functions defined by us from the ones defined by Opentrons’.<br>
| + | |
− | <br>
| + | |
− | </p>
| + | |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | |
− | While we wanted to establish Syn. elong. as a new chassis for the iGEM community and scientists we wanted to
| + | |
− | show the best conditions for cultivation and the best measuring method for our parts in UTEX 2973. Therefore
| + | |
− | we analyzed a big variety of cultivating conditions in measuring growth curves, tried to find a standard in
| + | |
− | light measurement, evaluated different reporters???, established a measurement method and compared it to a
| + | |
− | already known FACS measurement method (?).
| + | |
− | </p>
| + | |
− | <p style="text-align: justify; margin-bottom: 1em;">
| + | |
− | At the beginning of our project we faced the first question on how to cultivate UTEX at 1500 μE. [quelle].
| + | |
− | So we had to measure the light conditions in our incubators and while doing this simple task the first
| + | |
− | part of standardization began. We discovered that nearly every paper? is using different methods to measure
| + | |
− | their light conditions and that it is a really complex and important procedure. So we got in contact with
| + | |
− | cyano and light measurement experts [link IHP] to confront this problem and standardize it. In the following
| + | |
− | popup we show different ways of measurement, their (dis-)advantages and different results depending on the
| + | |
− | measuring instrument.<br>
| + | |
− | Not only the light intensity but also a variety of other cultivating parameters needed to be analyzed.
| + | |
− | In literature and while talking with different experts (IHP), we recognized that small deviations of these
| + | |
− | parameters had a huge impact on the growth speed of Synechococcus elongatus. While establishing UTEX 2973 as
| + | |
− | a new chassis we evaluated this impact on the growth speed and were able to show combinations of parameters
| + | |
− | that lead to the fastest growth speed.<br>
| + | |
− | Another aspect was measuring the expression and characterize our part. Different possibilities were
| + | |
− | discussed and after testing them we decided on two methods in our project (plate reader and FACs). One
| + | |
− | approach was to measure the fluorescence/luminescence with a plate reader [link part measurement]. Plate
| + | |
− | readers belong to standard equipment of every lab nowadays, and could deliver easy reproducible results.<br>
| + | |
− | The second way was to measure the fluorescence by FACS (Fluorescence-Activated Cell Sorting) [link facs]. In
| + | |
− | contrast to a platerader a FACs device delivers results with high accuracy by measuring every cell by its
| + | |
− | own(vielleicht erst spaeter FACS genau erklaeren aber nicht im abtract?). On the other side not
| + | |
− | every laboratory posses a FACs/device. So in the end we would like to offer a two method analyzed database
| + | |
− | from our crontructs for iGEM teams and research groups, who do not have access to a FACS and show the
| + | |
− | difference in measurement methods.<br>
| + | |
− | At the end of the project we were able to create a protocol how to handle Synechococcus elongatus UTEX 2973
| + | |
− | and make a contribution to the cyano community by establishing essential/fixed standards in measurement.
| + | |
− | </p>
| + | |
− | <figure align=center>
| + | |
− | <img style="height: 500px; width: 300px"
| + | |
− | src="https://static.igem.org/mediawiki/2019/b/bb/T--Marburg--opentrons_magnetic_module.JPG"
| + | |
− | alt="OT-2 left">
| + | |
− | <img style="height: 500px; width: 300px"
| + | |
− | src="https://static.igem.org/mediawiki/2019/3/30/T--Marburg--opentrons_shaker.JPG" alt="OT-2 right">
| + | |
− | <figcaption style="max-width: 1400px">
| + | |
− | Fig.2 - Single-Channel pipette, magnetic module and shaker in action while performing the plasmid
| + | |
− | purification.
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | <br>
| + | |
− | <p>
| + | |
− | Putting the pieces together, we were able to translate the manual plasmid purification protocol provided by Nans
| + | |
− | Bodet into an Opentrons protocol, being the very first of its kind. We pioneered a workflow for up to six
| + | |
− | samples with the p300 Single-Channel Electronic Pipette and a scaled-up version for up to 48 samples with the
| + | |
− | p300 8-Channel Electronic Pipette without having to intervene even once. This scalability provides important
| + | |
− | flexibility for various kinds of experiments.<br>
| + | |
− | <br>
| + | |
− | In our process of developing and running the protocol we determined some problems on increasing the yield of our
| + | |
− | plasmids. There was a large number of parameters that could be varied, changing the final concentration of the
| + | |
− | plasmids. For example, we realized that the duration of lysis is paramount for the yield and success of plasmid
| + | |
− | purification. Over-lysis will lead to a decrease in plasmid yield, whereas under-lysis will induce clumping of
| + | |
− | magnetic beads; thus failing the experiment. After a whole heap of plasmid purifications we managed to identify
| + | |
− | the most relevant parameters and improve the protocol in the best way possible.<br>
| + | |
− | <br>
| + | |
− | </p>
| + | |
− | <figure align=center>
| + | |
− | <img style="height: 700px; width: 600px"
| + | |
− | src="https://static.igem.org/mediawiki/2019/e/ea/T--Marburg--SingleChannelSetup.png" alt="OT-Layout left">
| + | |
− | <img style="height: 700px; width: 600px"
| + | |
− | src="https://static.igem.org/mediawiki/2019/d/df/T--Marburg--8channelSetup.png" alt="OT-Layout right">
| + | |
− | <figcaption style="max-width: 1400px">
| + | |
− | Fig.3 - Final setup for the automated plasmid purification workflows in the OT-2. The left picture shows the
| + | |
− | setup for the single channel workflow, the right picture for the 8-channel workflow.
| + | |
− | </figcaption>
| + | |
− | </figure>
| + | |
− | | + | |
− | <video src="https://static.igem.org/mediawiki/2019/c/c4/T--Marburg--PlasmidPurificationMarburg.mp4" controls
| + | |
− | poster="vorschaubild.jpg"></video>
| + | |
− | | + | |
− | <br>
| + | |
− | <br>
| + | |
− | </body> | + | |
| | | |
| </html> | | </html> |
| {{Marburg/footer}} | | {{Marburg/footer}} |