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| + | <LI> |
| + | <h3><a href="#PETModelling">2.1 Modeling PET degradation by <i>C. reinhardtii</i> using an optimized PETase</a></h3> |
| + | </LI> |
| + | <LI> |
| | | |
| <LI> | | <LI> |
− | <h3><a href="#GoldenGate">2.2 Transformation of PETase and MHETase into <i>C. reinhardtii</i> </a></h3> | + | <h3><a href="#Transformation">2.2 Transformation of PETase and MHETase into <i>C. reinhardtii</i> </a></h3> |
| </LI> | | </LI> |
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| + | |
| + | <div class="expandable-container"> |
| + | <div class="expandable-preview"> |
| + | <p class="medium-sized"> |
| + | A C. reinhardtii which expresses and secretes the enzymes PETase and MHETase could pose as a solution for the problem of micro-plastic polluted water. Nevertheless, the viability of PET-degradation by C. reinhardtii at a larger scale is yet unknown. |
| + | </p> |
| + | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> |
| + | </div> |
| + | <div class="expandable-more"> |
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
| + | |
| + | <div> |
| + | <p> |
| + | To assess the efficiency of PET-degradation by C. reinhardtii, a model of the expression, secretion and kinetics of the enzymes PETase and MHETase in C. reinhardtii was designed. The model can be seen <a href="https://2019.igem.org/Team:Humboldt_Berlin/Model">here</a>. |
| + | here. The goal of the model is to simulate the degradation of PET while taking into account the parameters of enzyme kinetics, expression rate, secretion rate and cultivation density. By varying the parameters, an approximation on PET degradation under various conditions can be made to examine what the appropriate parameters are for an optimal PET-degradation. To achieve this, the kinetics of MHETase and the optimized PETase (I179F) were taken from literature (Palm et al., 2019) (Ma et al., 2019). |
| + | </p> |
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| + | <div> |
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--modell_uebersicht.png" alt="PET degradation model" /> |
| + | |
| + | </div> |
| + | |
| + | </div> |
| + | |
| + | </div> |
| + | <div class="expandable-readall"> |
| + | <span class="readmore">Read more</span> |
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| + | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg"> |
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| </p> | | </p> |
| + | </div><div class="expandable-container"> |
| + | <div class="expandable-preview"> |
| + | <p class="medium-sized"> |
| + | Our first vision when we thought of our project in the beginning was to build a bioreactor in which Chlamy feels comfortable and is able to grow without interferences.<br> </br> |
| + | |
| + | Goals: increase protein yield, develop a method to set up cultivation tests with automated measurements easily and offer a cheap DIY reactor for cultivation of all photoautotrophic organisms<br> </br> |
| + | Iterations: changed from culture flask to flat panel reactor (experience gained at MINT), decreased culture volume, designed round flat panel to mix more thoroughly (expertise from Ullman’s algae farm), increased wavelength options for cultivation of various organisms<br> </br> |
| + | |
| + | </p> |
| + | <img src="https://static.igem.org/mediawiki/2019/1/1b/T--Humboldt_Berlin--designfig1.png" alt="Overview of the hierarchical and modular cloning system" /> |
| + | </div> |
| + | <div class="expandable-more"> |
| + | <div class="two-columns block-text medium-sized not-centered no-margin-top" style="margin-bottom:0px"> |
| + | |
| + | <div> |
| + | <p> |
| + | Along the way of performing our experiments and making our parts, we recognized that there is one big bottleneck in growing not only C. reinhardtii but also other photosynthetic organisms: lack of an affordable and reproducible cultivation setup in a laboratory scale. We therefore decided to address this problem by designing and constructing an open source platform that is capable of growing phototrophic organisms in turbidostat mode, not only monitoring growth but also making it possible to adjust environmental conditions.<br> </br> |
| + | |
| + | To help us in the process of design, we asked several sources with experience and knowledge in algae cultivation on different scales to get a first overview of problems and demands of cultivation (Joerg Ullmann, Gunnar Muehlstaedt, Ralf Steuer ). With this help and knowledge gathered from modeling, we were able to pin down necessary functions our platform has to fulfill and therefore components it has to be built of. We always keep one function important to the Synbio community in the back of our head: The ability to perform many experiments and/or screening events at once. This means that a general demand for our setup is to be easily affordable, reproducible and manufacturable with as little effort as possible. <br> </br> |
| + | |
| + | Taking these demands and information into account we decided on the following functions and components performing them: |
| + | |
| + | Different types of cultivation vessels with self built flat panel for higher density; |
| + | Photometer to measure optical density; |
| + | Regulation of air supply and mixing of the culture; |
| + | Photon source e.g. LED; |
| + | Temperature Measurement and control; |
| + | Control of pumps in a Turbidostat setup |
| + | |
| + | |
| + | As every single component gives rise to necessary tests for assessing its function (such as calibration of the photometer and pumps), details of experiments performed are depicted on the bioreactor subpage. |
| + | |
| + | We will create a library of step by step tutorials showing how to build all of these different functions and make it possible and motivate other iGEM participants and scientists to engage in do it yourself (DIY) setups and electronics. |
| + | |
| + | To perform our needed functions, we will build a small low-cost DIY photometer which can measure the optical density (OD) of C. reinhardtii at 680 nm or any other wavelength in the visible spectrum. The photometer is designed to not only work inside our cultivation setup, but can also be used as a stand-alone solution for OD measurement. |
| + | |
| + | Given the possibility of using several different cultivation vessels, we designed our setup to hold standard Schott 45 flasks, cell culture flasks with 3D printed screw-tops and flat panels designed by ourselves, while being able to hold any other cultivation vessel with the appropriate dimensions. |
| + | |
| + | |
| + | Gas supply and mixing of a culture are important factors to maintain a constant cell growth and, therefore, have to be monitored closely. We tried to design a solution with a rotameter which is easy to control to achieve a high chance of reproducibility. It is also possible to measure and regulate the temperature of the cultivation vessel to obtain reliable data. |
| + | |
| + | For temperature control, we decided to immerse our cultivation vessels in a water bath where temperature can be measured and held constant through a heating element. |
| + | |
| + | Because in a photobioreactor setup the photon source is probably the most expensive and complicated part, we decided to build in a RGBW light source with an intensity of at least 300 µE to make light-dependent growth at high densities possible. |
| + | |
| + | To be able to reproduce results from Flux Balance Analysis and perform other experiments which demand steady state, we wanted to include a turbidostat-function in our setup by installing two pumps that can hold OD of the culture constant. Removed culture can be used for transformation experiments or for screening. |
| + | |
| + | </p> |
| + | |
| + | </div> |
| + | |
| + | <!--- IMAGE ---> |
| + | <div> |
| + | <img class="is-revealing" src="https://static.igem.org/mediawiki/2019/3/32/T--Humboldt_Berlin--modell_uebersicht.png" alt="PET degradation model" /> |
| + | |
| + | </div> |
| + | |
| + | </div> |
| + | |
| + | </div> |
| + | <div class="expandable-readall"> |
| + | <span class="readmore">Read more</span> |
| + | <span class="readless">Read less</span> |
| + | <img src="https://static.igem.org/mediawiki/2019/3/3e/T--Humboldt_Berlin--ArrowDown.jpg"> |
| + | </div> |
| </div> | | </div> |
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| <!--- IMAGE ---> | | <!--- IMAGE ---> |