Team:TU-Munich/HumanPractice/Education

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Introducing Synthetic Biology Kits at educational institutions

What is better than teaching one class about SynBio?
Enabling many teachers to complete this task!

"If you want to build a ship, do not drum up people together to collect wood and do not assign them tasks and work, but rather teach them to long for the endless immensity of the sea."

- Antoine de Saint-Exupéry

Introduction

Figure 1: Prepared Educational-Kits, ready for delivery!

Following this famous statement in pedagogics, we realized the importance to inspire and teach students in educational institutions to long for the endless immenseness of Synthetic Biology. We want to make it possible for disciples to take their first steps in the enormously evolving field of Synthetic Biology and let them take a breath of this subject, which will change the world in the near future due to its unlimited fields of application.

The idea is to send an "Educational-Kit" with all required reagents and lab-protocols to educational institutions. Based on the experiments we designed we'd like to inspire young prospective people to think about a promising career in science. Our experiments are styled constitutive, based on every previously performed experiment, so every student, independent of training level, is able to understand and take notice of the resulting phenomena enabled by Synthetic Biology.

The Educational-Kit

The reagents, which are necessary for the realization of the experiments are all found in the kit, except for competent cells, which have to be stored at -20°C. It was set much value on the autonomous preparation of the final reagents, so nearly all reagents will be sent in their blank shape. Our aim is teaching the students all, or at least most, of the necessary steps of the designed experiments during the execution. The kit consists of the following reagents:

**Table 1:** Contents of the educational kit
Reagent	Amount of the Reagent	Is it existent in the kit?
LB-Medium	Reagents for about 3 L are in the kit	Yes
"Banana-Odor"-Plasmid	100 µl (10 ng/µl)	Yes
"RFP-Generator"-Plasmid	100 µl (10 ng/µl)	Yes
"Luciferase"-Plasmid	100 µl (10 ng/µl)	Yes
Isoamylalcohol (>98 %)	10 ml	Yes
Agar plates (Resistance: Kanamycin)	4 Plates	Yes
Agar plates (Resistance: Chloramphenicol)	2	Yes
L-Arabinose (500 mM)	3.76 g (50 ml)	Yes
Kanamycin	25 ml	Yes
Chloramphenicol	25 ml	Yes
Competent Cells	6 Tubes (Aliquotvolume: 150 µl)	No

As you may have noticed, we provide three different plasmids, which are used separately in each designed experiment. After the experiments have been performed, disciples should have experienced the whole way from transformation, over cultivating cells, inducing the production of proteins. In the end they should also have observed the conversion of a substrate by the produced enzyme.

Experiment Number 1: Bacteria in Red

Figure 2: Results of Experiment Number 1

The aim of the first experiment is to transform the [http://parts.igem.org/Part:BBa_K801100 "RFP-Generator"-Plasmid] into Escherichia coli, which contains genes, coding for Chloramphenicol resistance, plus a red fluorophor. Then they should be plated on Chloramphenicol-Agarplates. The Agarplates, provided with the antibiotica Chloramphenicol, trigger a selection process, whose aim is to kill all bacteria which have not incorporated the "RFP-Generator"-Plasmid. The expression rate of the red fluorophor is controlled by a constitutive promoter, so the Red Fluorescent Protein is expressed continuously. After just about one day of incubation time, the students can notice a change in color of the bacteria from ocher to red with the naked eye.

The Biobrick we used to realize this experiment was firstly designed by the iGEM Team of Groningen in 2010 to simplify the cloning step by detecting internalized plasmids, which self-circularized without integration of the desired insert. Bacteria, which internalize self-circularized Plasmids will produce red colonies, so these undesired bacterial colonies can be avoided in the next steps of the experiment, to be sure of using the right construct.

Experiment Number 2: Glowing Bacteria Lamp

Figure 3: Structure of the Luciferase

In the second experiment students should perform the transformation, which they learned in experiment one, with the "Luciferase"-Plasmid. The Plasmid encodes genes for Kanamycin resistance and a lux - gene cassette containing a protein-fluorophor called Luciferase. Another very important point is that the lux-gene is subordinated to a L-Arabinose Operon. After the transformation of the DNA-Plasmid has taken place, students should learn how to cultivate bacteria in liquid LB-Medium and how to induce protein expression by using L-Arabinose as an inductor of the L-Arabinose promoter. The glow of the Luciferase can already be observed after a few hours.

Experiment Number 3: Banana Odor Generator

Figure 3: Results of Experiment three

In the third experiment, all aspects learned in the previous experiments come together. The [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] encodes an enzyme called Alcohol-Acetyltransferase I. This enzyme is able to convert Isoamylalcohol into Isoamylacetat, which spreads the odor of banana. Additionally the plasmid harbors a resistance gene for Kanamycin and an inducible L-Arabinose promoter, which controls the expressions rate of the Alcohol-Acetyltransferase I enzyme. The students should transform the [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] into Escherichia coli, cultivate the successfully transformed bacteria in a larger scale and in the end, induce the protein expression by using L-Arabinose. In the last step, the students should add the substrate Isoamylalcohol to the cell culture, so the produced enzyme can transform it into Isoamylacetat.

The Biobrick, which spreads the odor of banana, after transforming its substrate, was first established by the [http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team of the Massachusetts Institute of Technology in 2006]. Their aim was to produce different compounds in Escherichia coli that smell fragrant. Another very interesting application of the "Banana-Odor" Biobrick is pursued by the iGEM Team of Queens in 2013. This team tries to neutralize foot odor by creating a skin creme containing bacteria with a genetically engineered metabolic pathway to neutralize the volatile compounds that cause these smells. This pathway begins with the uptake of isovaleric acid, a known mosquito semiochemical that is present in foot odor, and converts it into banana smell. By establishing this skin creme, it should be possible to prevent people from being attacked by mosquitos and saved from typical alienable diseases mosquitos spread.

Target Audience

The target audience for our kit are secondary schools with focus on a biotechnical educational pathway, so all students have access to the required laboratory equipment for the experiments. Before sending the kits to the schools, we tested them in detail - with success. All schools we contacted to spread our Synthetic Biology kits are very enthusiastic concerning our idea of supporting young prospective scientists, so they are very excited to receive and use our kit!

Availability of the School-kit

We appreciate other interested schools in trying our “School kits”. Do not hesitate contacting us, so we can supply you with our forward-looking Synthetic Biology “School-Kit”!

You can send requests to: igem@wzw.tum.de

References

http://www.ncbi.nlm.nih.gov/pubmed/22368493 Close D et al., 2012 Close D, Xu T, Smartt A, Rogers A, Crossley R, Price S, Ripp S, Sayler G (2012). The evolution of the bacterial luciferase gene cassette (lux) as a real-time bioreporter. Sensors, 12(1):732-52.

http://parts.igem.org/Part:BBa_K801100 BBa_K801100: "RFP-Generator"-Biobrick

http://parts.igem.org/Part:BBa_J45014 BBa_J45014:"Banana-Odor"-Biobrick

[iGEM Team Groningen 2010]

http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team Massachusetts Institute of Technology 2006

[iGEM Team Queens 2013]

@@ Line 95: / Line 95: @@
 ==Experiment Number 3: Banana Odor Generator==
 [[File:SchmolkerMollath.png|thumb|right|400px|'''Figure 3:''' Results of Experiment three]]
-In the third experiment, all aspects learned in the previous experiments come together. The [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] encodes an enzyme called Alcohol-Acetyltransferase I. This enzyme is able to convert Isoamylalcohol into Isoamylacetat, which spreads the odor of banana. Additionally the plasmid harbors a resistance gene for Kanamycin and an inducible L-Arabinose promotor, which controls the expressions rate of the Alcohol-Acetyltransferase I enzyme. The students should transform the [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] into ''Escherichia coli'', cultivate the successfully transformed bacteria in a larger scale and in the end, induce the protein expression by using L-Arabinose. In the last step, the students should add the substrate Isoamylalcohol to the cell culture, so the produced enzyme can transform it into Isoamylacetat.
+In the third experiment, all aspects learned in the previous experiments come together. The [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] encodes an enzyme called Alcohol-Acetyltransferase I. This enzyme is able to convert Isoamylalcohol into Isoamylacetat, which spreads the odor of banana. Additionally the plasmid harbors a resistance gene for Kanamycin and an inducible L-Arabinose promoter, which controls the expressions rate of the Alcohol-Acetyltransferase I enzyme. The students should transform the [http://parts.igem.org/Part:BBa_J45014 "Banana-Odor"-Plasmid] into ''Escherichia coli'', cultivate the successfully transformed bacteria in a larger scale and in the end, induce the protein expression by using L-Arabinose. In the last step, the students should add the substrate Isoamylalcohol to the cell culture, so the produced enzyme can transform it into Isoamylacetat.
-The Biobrick, which spreads the odor of banana, after transforming its substrate, was first established by the [http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team of the Massachusetts Institute of Technology in 2006]. Their aim was to produce different compounds in ''Escherichia coli'' that smell fragrant. Another very interesting application of the "Banana-Odor" Biobrick is pursued by the [https://2013.igem.org/Team:Queens_Canada/Project/Repel iGEM Team of Queens in 2013]. This team tries to neutralize foot odour by creating a skin creme containing bacteria with a genetical engineered metabolic pathway to neutralize the volatile compounds that cause these smells. This pathway begins with the uptake of isovaleric acid, a known mosquito semiochemical that is present is foot odour, and converts it into banana smell. By establishing this skin creme, it should be possible to prevent people from being attacked by mosquitos and saved from typical alienable diseases mosquitos spread.
+The Biobrick, which spreads the odor of banana, after transforming its substrate, was first established by the [http://openwetware.org/wiki/IGEM:MIT/2006 iGEM Team of the Massachusetts Institute of Technology in 2006]. Their aim was to produce different compounds in ''Escherichia coli'' that smell fragrant. Another very interesting application of the "Banana-Odor" Biobrick is pursued by the [https://2013.igem.org/Team:Queens_Canada/Project/Repel iGEM Team of Queens in 2013]. This team tries to neutralize foot odor by creating a skin creme containing bacteria with a genetically engineered metabolic pathway to neutralize the volatile compounds that cause these smells. This pathway begins with the uptake of isovaleric acid, a known mosquito semiochemical that is present in foot odor, and converts it into banana smell. By establishing this skin creme, it should be possible to prevent people from being attacked by mosquitos and saved from typical alienable diseases mosquitos spread.
 ==Target Audience==