Difference between revisions of "Team:Humboldt Berlin/Hardware"

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                     <img src="https://static.igem.org/mediawiki/2019/4/4d/T--Humboldt_Berlin--Cultivation_chamberbro.jpeg" alt="Flat panels inside the bioreactor" />
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                        The LED panel of the openPBR is designed to give you full control of both color and intensity of the light source powered by 250 single RGB LEDs and 250 white LEDs with a total light intensity of up to 300 µE. Having a constant and reproducible light source in a photobioreactor setup is one of the most difficult tasks to achieve. To learn how to build and control your own please visit our step-by-step tutorial.
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To get an impression of how to set up the relative voltage in the OBP-software to get the desired light intensity, we prepared an light intensity adjustment curve.We measured the light intensity at 24 different positions above the LED panel, each with the relative voltage set to 15, 140 und 225. Using this values we calculated linear fit which can be used to predict the light intensity at a given relative voltage.  
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                  To get an impression of how to set up the relative voltage in the OBP-software to get the desired light intensity we developed a light intensity adjustment curve. We measured the light intensity at 24 different positions above the LED panel, each with the relative voltage set to 15, 140 and 225. Using these values we calculated a linear fit, which can be used to predict the light intensity at any given relative voltage.
 
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Revision as of 11:32, 21 October 2019

MathJax example

plasmid

Hardware

OPEN PBR LOGO

Introduction

Designing the bioreactor

Overview of single components

Assembly instructions

Introduction

The openPBR is a low-cost, easy to assemble and reliable option to cultivate phototrophic organisms. We have developed it as an open-source platform to operate as you need it. It comes in several setups, from nine cultivation vessels to three flat panels with a minimum width of 15 mm and up to a volume of 190 ml. It provides users with the ability to measure optical density at wavelengths from 400 nm to 800 nm, online and provides a .csv file output. All parts are openly available and even gas supply is exactly adjustable through a rotameter, depending on the needs of your organism and setup. We want to offer scientists a simple and versatile cultivation platform.

    Overview of single components

  • Sensors

  • Illumination

  • Gas mixing and pumps

  • Cultivation chamber

  • Casing

Our Open PBR Electronic Gas Supply LED-Panel Pumps Cultivation Chamber Sensors Casing
Klick on part-labels for more information

Cultivation Chamber

The openPBR can be used with several different cultivation vessels. Originally designed to hold three 95 ml, fully autoclavable, flat panels, with interchangeable rings from 15 mm (95 ml) up to 30 mm width (190 ml). For growth-comparison studies, the openPBR can be set up with up to nine hybridization flasks with standard SCHOTT-45 cups. Even the use of cell-culture flasks is possible. For this use-case we 3D-printed autoclavable cups to connect them with hoses for classical cell-culture flasks.

Cultivation chamber_menübild
Flat panels inside the bioreactor

Replacement rings for flatpanel
Fig. 1. figurecaption bold. figure caption description
Cell flasks for the bioreactor

3D-printed screw tap
Fig. 1. figurecaption bold. figure caption description
Read more Read less

Illumination

The LED panel of the openPBR is designed to give you full control of both color and intensity of the light source powered by 250 single RGB LEDs and 250 white LEDs with a total light intensity of up to 300 µE. Having a constant and reproducible light source in a photobioreactor setup is one of the most difficult tasks to achieve. To learn how to build and control your own please visit our step-by-step tutorial.

LED panel

To get an impression of how to set up the relative voltage in the OBP-software to get the desired light intensity we developed a light intensity adjustment curve. We measured the light intensity at 24 different positions above the LED panel, each with the relative voltage set to 15, 140 and 225. Using these values we calculated a linear fit, which can be used to predict the light intensity at any given relative voltage.


Fig. 1. bold figure caption. figure caption description
Read more Read less

Gas supply

For operating our culture in chemostat mode, we need pumps for transportation of medium in and out of our culture vessel as well as for mixing of gas into the culture for growth. This chapter aims to explain important parameters while choosing a pump for the setup.

Overview of the hierarchical and modular cloning system

WHAAT?


Our Gas and mixing pumps

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

Cultivation chamber

Because of Human Practices and Modeling, we decided to build a flat-panel cultivation vessel for our culture. Here we briefly explain our thoughts while building our own vessel and choosing vessels that fit our cultivation setup.

Overview of the hierarchical and modular cloning system

WHAAT?


flat-panel cultivation vessel

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

empty cltivation vessel with tubes

Fig. 2. Blablabla.

Figcaption.
Read more Read less

Casing

We shortly explain necessary properties of the case that holds our cultivation setup.

Overview of the hierarchical and modular cloning system

WHAAT?


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

Electronic

In this chapter, we will show you the sensors we are implementing in our setup. We build our own photometer and are using a prebuilt thermometer for temperature monitoring.

Overview of the hierarchical and modular cloning system

This already defines the components we need for measuring amount of algae cells, an LED that emits light ( \(I_0)\) ), a sensor that detects light after passage through the vessel and the vessel itself. It is important to measure under the same conditions every time, because otherwise \( I_0 \) can vary due to scattering of light at the vessel. Linearer Opt 101 in wichtigem Bereich (PAR)


The light detectors are inside of the metal strip

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

    Designing the bioreactor

  • Chemostat equation

  • Light Gradient

  • OD Measurement

Bringing Chlamy to iGEM

Chemostat Equation

This chapter gives an overview over important parameters for cultivation resulting from the general chemostat equation.

Overview of the hierarchical and modular cloning system

The chemostat equation which we treated as a part of modeling provides us with an overview of general parameters important to a microbial culture. If we want to cultivate in turbidostat-mode, we need to remove liquid from the culture continuously. This removing is quantified through dilution rate \( D \): \begin{equation} D = \frac{1}{V_{c} \cdot \frac{\mathrm{d}}{\mathrm{d}t} V_{in} } \end{equation} \begin{array}{r l} V_c:& \text{Volume of culture in} mL \\ V_{in}: & \text{Volume of added medium in} mL \end{array} This equation shows us how


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

Light Gradient

An important aspect of photosynthetic growth is illumination. Read about the light environment in a cultivation vessel here.

Overview of the hierarchical and modular cloning system

WHAAT?


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

OD Measurement

With the same equation describing the light gradient inside the culture, we can define a relationship to measure cell density and other parameters.

Overview of the hierarchical and modular cloning system

Measurement of \(OD\) is based on the Beer-Lambert law (see our model for further information). From this law, \(OD\) is defined as: \begin{equation} OD = - log(\frac{I(\lambda)}{I_0(\lambda)}) = \epsilon_X \cdot c_X \cdot d \end{equation} \newcommand\T{\Rule{0pt}{1em}{.3em}} \begin{array}{r l} I_0(\lambda, t): & \text{light intensity upon entrance into vessel in} \: \frac{\mu mol}{m^2 \cdot s} \\ \epsilon_X: & \text{light attenuation coefficent for algae in} \: \frac{L}{mol \cdot cm} \\ bg: & \text{background turbidity} \: \frac{1}{cm} \\ d: & \text{light path in} cm \end{array}
Generally speaking, realization of the setup should make the variables in the equation as indifferent to parameters that have an impact on measurement as possible.


Cultivation vessel with chlamydomonas Reinhaditii

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

    Assembly Instructions

  • Github Project

  • Tools

  • PDF Instructions

This chapter provides you with everything you need to rebuild our setup on your own - including software, a list of used parts and assembly instructions.

Github Project

Visit our Github page, where assembly instructions, list of parts and software are provided together and are kept up to date!

Overview of the hierarchical and modular cloning system
Read more Read less

Tools

Container preview

Overview of the hierarchical and modular cloning system

WHAAT?


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

PDF Instructions

Look at our PDF instructions where we provide you with a detailed step-by-step tutorial to build your own OPEN PBR!

Overview of the hierarchical and modular cloning system

WHAAT?


Overview of the hierarchical and modular cloning system

Fig. 1. Universal MoClo fusion sites.

Figcap

WHAAAT?

WHAAAT?

WHAAAT?

cloning strategy

Fig. 2. Blablabla.

Figcaption.
Read more Read less

Crozet, P., Navarro, F. J., Willmund, F., Mehrshahi, P., Bakowski, K., Lauersen, K. J., ... Lemaire, S. D. (2018). Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii. ACS Synthetic Biology, 7(9), 2074-2086. Retrieved from https://doi.org/10.1021/acssynbio.8b00251. doi:10.1021/acssynbio.8b00251

Ebrahim, A., Lerman, J. A., Palsson, B. O., & Hyduke, D. R. (2013). COBRApy: COnstraints-Based Reconstruction and Analysis for Python. BMC Systems Biology, 7(1), 74. https://doi.org/10.1186/1752-0509-7-74

Engler, C., Kandzia, R. & Marillonnet, S. (2008). A One Pot, One Step, Precision Cloning Method with High Throughput Capability. PLOS ONE, 3(11), e3647. Retrieved from https://doi.org/10.1371/journal.pone.0003647. doi:10.1371/journal.pone.0003647

Greiner, A., Kelterborn, S., Evers, H., Kreimer, G., Sizova, I. & Hegemann, P. (2017). Targeting of Photoreceptor Genes in Chlamydomonas reinhardtii via Zinc-Finger Nucleases and CRISPR/Cas9. The Plant Cell. 29. tpc.00659.2017. 10.1105/tpc.17.00659.

Imam, S. , Schäuble, S. , Valenzuela, J. , López García de Lomana, A. , Carter, W. , Price, N. D. and Baliga, N. S. (2015), A refined genome‐scale reconstruction of Chlamydomonas metabolism provides a platform for systems‐level analyses. Plant J, 84: 1239-1256. doi:10.1111/tpj.13059

Kelterborn, S., Boehning, F., Evers, H., Sizova, I., Baidukova, O., & Hegemann, P. (2019). Gene editing in green alga Chlamydomonas reinhardtii via CRISPR-Cas9 ribonucleoproteins. Unpublished work.

Kliphuis, A. M. J., Klok, A. J., Martens, D. E., Lamers, P. P., Janssen, M., & Wijffels, R. H. (2012). Metabolic modeling of Chlamydomonas reinhardtii: Energy requirements for photoautotrophic growth and maintenance. Journal of Applied Phycology, 24(2), 253–266. https://doi.org/10.1007/s10811-011-9674-3

Loera‐Quezada, M. M., Leyva‐González, M. A., Velázquez‐Juárez, G. , Sanchez‐Calderón, L. , Do Nascimento, M. , López‐Arredondo, D. and Herrera‐Estrella, L. (2016), A novel genetic engineering platform for the effective management of biological contaminants for the production of microalgae. Plant Biotechnol J, 14: 2066-2076. doi:10.1111/pbi.12564

Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., ... & Yuan, Y. (2018). Enhanced poly (ethylene terephthalate) hydrolase activity by protein engineering. Engineering, 4(6), 888-893. Mali, P., Yang, L., Esvelt, K. M., Aach, J., Guell, M., DiCarlo, J. E., Norville, J. E., and Church, G. M. “RNA-Guided Human Genome Engineering via Cas9,” Science, vol. 339, pp. 823–826, feb 2013.

Merchant, S. S., Prochnik, S. E., Vallon, O., Harris, E. H., Karpowicz, S. J., Witman, G. B., … Grossman, A. R. (2007). The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science (New York, N.Y.), 318(5848), 245–250. doi:10.1126/science.1143609

Patron, N. J., Orzaez, D., Marillonnet, S., Warzecha, H., Matthewman, C., Youles, M., . . . Haseloff, J. (2015). Standards for plant synthetic biology: a common syntax for exchange of DNA parts. New Phytologist, 208(1), 13-19. Retrieved from https://doi.org/10.1111/nph.13532. doi:10.1111/nph.13532

Weber, E., Engler, C., Gruetzner, R., Werner, S., & Marillonnet, S. (2011). A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS ONE, 6(2), e16765. Retrieved from https://doi.org/10.1371/journal.pone.0016765. doi:10.1371/journal.pone.0016765

Palm, G. J., Reisky, L., Böttcher, D., Müller, H., Michels, E. A., Walczak, M. C., ... & Weber, G. (2019). Structure of the plastic-degrading Ideonella sakaiensis MHETase bound to a substrate. Nature communications, 10(1), 1717.

Strenkert, S., Schmollinger, S., Gallaher, S. D., Salomé, P. A., Purvine, S. O., Nicora, C. D., Mettler-Altmann, T., Soubeyrand, E., Weber, A. P. M., Lipton, M. S., Basset, G. J., Merchant, S. S. Proceedings of the National Academy of Sciences Feb 2019, 116 (6) 2374-2383; DOI:10.1073/pnas.1815238116

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