Team:Heidelberg/Sandbox

iGEM Team Heidelberg 2015

• Achievements
• Results
• Project
• Software
• Notebook
• Parts
• Team
• Modeling
• Outreach

Short introduction

“The sea is everything. […] The sea is the vast reservoir of Nature. The globe began with sea, so to speak”, Nemo said while the “Nautilus” was cruising with a school of hammerhead sharks deep beneath the waves.

And the captain was right. Deep in the ocean billions of years ago the miracle of nature took place as a pool of small molecules evolved to self-replicating lifeforms. The flagship role in this development was probably taken by the most versatile class of molecules in the history of life: RNA.

Seemingly random nucleotides happened to be in the right order to form the first biocatalysts that made life on the blue planet possible. Today we know those miracles of nature as ribozymes. Inspired by this, humanity took evolution into their own hands to create aptamers – nucleic acids capable of encaging molecules. This allows for the detection of virtually anything. Still this process has been tedious and time consuming much like fishing with a rod in an ocean. We want to revolutionize this former evolutionary process and want to make it swift like a shark tracking down its prey.

Yet to really bring out the strengths of these simple yet powerful molecules just comprised of A, U, C and G we want to combine aptamers and ribozymes to create a toolset for the synthetic biologist to create allosteric ribozymes able to sense a variety of molecules. Therefore, we hope to introduce the true origins of life and the capabilities of functional RNA to iGEM.

Join us as we sail forth into new waters of synthetic biology.

Further content

Test

Test

Notebook

Supporting iGEM Team Paris Bettencourt

iGEM Paris Bettencourt has set up a visual databse that should help various iGEM teams collaborate and find common ground. In that database all teams can enter techniques, keywords, organism and many more thev are knowdledgeable in or have been working on. As common dots are connected automatically, it's easy so see, whom to ask for a collaboration.
Though we came a little late to the party, we really loved the idea and hoped, that the database has helped various teams!

"Rhizi is an open-sourced web of knowledge where nodes are sources of information (e.g. scientific articles, questions, blog posts) and edges are the links between them. The goal of Rhizi is to create information rich links between knowledge, open questions and ideas, through encouraging users to vote, comment, and create new relationships."

http://cri-paris.org/research/rhizi/

Collecting impressions from the community

It’s done. We’ve finally decided upon a logo. It’s beautiful, it’s blue, it’s fishy *wink*. The next step was to choose a color scheme and a general style for the wiki. As we feared that our own ideas might be too similar we thought of asking a broader public how they would design a website based on our logo. In order to do so, we headed to the ’Neckarwiese’. During summer, a big part of Heidelbergs population decideds to go there and enjoy the sun, have a BBQ or just relax – so we caught them off guard.

We have had prepared a little presentatio with which we introduced them to iGEM and Synthetic Biology. Afterwards we explained them what we planned on doing, showed them our logo and asked, how they would design a website, based on what they’ve just heard. The general consens was to go for a blue theme, maybe add a little algae green and generally stick to maritime iconography. Unexpectedly we actually met someone who knew his stuff: Natalie, a student of the history of arts from Berlin. She gave us useful advice on how play with the different colors, what to avoid and even added a little touch of history to it. Lucky catch!

With fresh ideas we headed back to the lab and continued our experiments, being envious of all the people that could continue to sleep in the sun.

We’ve been lucky and actually met someone knowledgable in arts. Her name is Nati and she studies the history of art in Berlin.

Panel Discussion

Synthetic biology - engineering life with its most fundamental units by using DNA BioBricks and other modularly combinable parts, has a potential beyond scope and can improve the quality of life for everyone and mankind as a whole. The ultimate goal of researchers in synthetic biology is not only the understanding of life itself and how it functions, but applying the acquired knowledge to make a change within their community.

To investigate the current state of the research and its acceptance in society, we talked to scientists and asked them how their work has influenced their community. Nonetheless, it is of absolute necessity to include the entire society. We decided to do this by organizing a panel discussion evening dedicated to the topic 'Synthetic biology - Bricks for a healthy life?', i.e. synthetic biology in medicine.

As research in general and especially synthetic biology relies on a community, on interaction between researchers and the exchange of ideas and expertise, we asked experts and researchers from different fields to join us for this evening and we distributed flyers and placards to invite the broad society. However, we wanted to go a step further: The panel discussion was not limited to the audience in Heidelberg, it was simultaneously translated from German to English and broadcasted it via a live stream.

Despite the great enhancements synthetic biology can achieve, engineering with building blocks that are so close to the basic principles of life itself comes with a range of ethical questions and security precautions to consider. We see it as our immanent responsibility as iGEM team to address these questions and to take concerns very seriously. Hence, we invited Prof. Dr. Axel Bauer, who has been member of the German ethics council for several years and Dr. Joachim Boldt who works as assessor for the German ethics commitee for ethical implications of synthetic biology. Both of them are highly involved in the field of medical ethics also due to being professor for this subject. In order to review the safety concerns, we were very glad to have Dr. Harald König, who works at the Institute for Technology Assessment and System Analysis, as our guest.

Different experts were invited to an interdisciplinary talk evening

Politics and law play a very big role when discussing the application of synthetic biology in real life. Researchers need to obey the juridical boundaries and on the contrary the legislature has to react to novel developments and find a compromise between many different opinions, some being more conservative, others more progressive. To reflect this interweaving, we additionally invited local politicians, such as Prof. Dr. Nicole Marmé who is member of the city council of Heidelberg. Dr. Stephan Brandt, chairman of the department for Biotechnological Innovation, Nanotechnology and Genetic Engineering, investigates how laws need to be adapted to the most recent findings in synthetic biology and genetics and we are very glad he joined us for this evening. Finally, the topic that stands in the center of this evening is, after all, research in synthetic biology. For that reason, we asked Dr. Dirk Grimm, who works on the CRISPR-Cas system and who knows the cutting edge developments to be the scientific representative in the panel.

Jasmin and Max gave a great introduction to synthetic biology in medicine

After a lot of planning, organization and set up of all the required technical equipment (thanks again to Dr. Jens Wagner from the Physics Department), the discussion evening could start with a brief introduction to the topic given by Jasmin and Max from our team. As we wanted the main part of the evening to be an open discussion, we deliberately made the introduction very compact. For the remaining 1.5 hours of the evening, Tim navigated our guests, as well as the audience through the discussion.

The involvement of the audience was amazing, proving that this topic is indeed highly interesting to a large part of the society. Most eminently, we were very happy that additional to the approximately 100 guests that joined us physically in our institute, we had almost 400 viewers online, among them also other iGEM teams, such as iGEM Team Cambrige (link). Besides watching, our followers on twitter were also very engaged in asking questions that were then addressed by Tim and the invited experts.

Luckily, many people came to our talk evening. Even more joined us online via our simultaneously translated live stream and asked questions with #askigemheidelberg

The topics discussed ranged from green to red biotechnology and were contemplated in a highly interdisciplinary way (with the focus on medical applications nonetheless). Besides, and in correspondence to the initialization of the “Community lab” track in iGEM, we addressed biohacking and the implications of it on society, the scientific community and the communication between the two entities. Question were asked about ethical problems and implications of in vivo and in vitro technologies, but also about dreams and wishes of the scientists regarding future developments in this field. The question iGEM team Cambridge nicely summarizes the last part of the discussion: “How can synthetic biologists better communicate their research to the public?” This includes the role of politicians and law makers, as well as the responsibility of everyone who is involved in research to put a focus on the outreach and the interaction, not only with the scientific community, but also with the broader public.

So far, many iGEM teams have organized discussion evenings and invited people from the broad society to join an interdisciplinary evening. This approach is great and helps a lot to improve the communication between scientists and the public. Nonetheless, there are still barriers to overcome:

• The interested population of one city is not representing the entire society. Hence, we decided to provide the opportunity to join us online via live stream. This should not be limited to watching the discussion passively, that is why everyone could ask questions via twitter by #askigemheidelberg. These questions were then shown in the discussion, so that our invited guests could reply or reflect on them.
• The lingua franca in research is English, however not everybody is capable of speaking English fluently. Therefore, we deliberately chose German as the language the discussion was held in. This way, everyone who was interested had the possibility to follow. In order to keep the event international and also understandable for those who watched online, we translated the event simultaneously to English.

week number 38

▼2015-09-14 Test bitte löschen

10 x <i>in vitro</i> transcription buffer

50 mM Tris ph 7.5, 100 mM NaCl, 20 mM MgCl₂, 0,01 % SDS
 

week number 37

▼2015-09-10 DNAzyme Activity: DNAzyme with ATP aptamer and calculated Kanamycin aptamer

Samples:

• 10-23 DNAzyme: xxfs032xx
• 7-18 DNAzyme: xxfs033xx
• 10-23 DNAzyme with ATP aptamer with linker: xxfs019xx
• 7-18 DNAzyme with ATP aptamer with linker: xxfs027xx
• 10-23 DNAzyme with ATP aptamer A: xxfs017xx, B: xxfs018xx
• 7-18 DNAzyme with ATP aptamer A: xxfs025, B: xxfs026xx
• 10-23 DNAzyme with calculated Kan aptamer: xxfs034xx
• 7-18 DNAzyme with calculated Kan aptamer candidate I: xxfs035xx
• 7-18 DNAzyme with calculated Kan aptamer candidate II: xxfs036xx
• 7-18 DNAzyme with calculated Kan aptamer candidate III: xxfs037xx

Stock solutions and conditions:

	cStock	cFinal
Tris HCl ph 7.5	1 M	50 mM
DNAzyme (A)	10 µM	500 nM
DNAzyme B	10 µM	500 nM
Substrate	1 µM	200 nM
NaCl	1 M	100 mM
MgCl2	1 M	20 mM
SDS	20 %	0,01 %
Adenosine in H2O:DMSO 1:2	33 mM	5 mM
H2O		ad 25 µL

 

Pipetting scheme:

#			Tris HCl ph 7.5	DNAzyme A	DNAzyme B	Substrate	NaCl	MgCl2	SDS	Adenosine	H2O	Final
1	FS032	10-23D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
2	FS033	7-18D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
3	FS019	10-23DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
4	FS027	7-18DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	3,75	9,50	25,00
5	FS017+18	10-23D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	3,75	3,25	25,00
6	FS025+26	7-18D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	3,75	3,25	25,00
7	FS032	10-23D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
8	FS033	7-18D	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
9	FS019	10-23DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
10	FS027	7-18DmLink	1,25	1,25	0,00	5,00	2,50	0,50	1,25	0,00	13,25	25,00
11	FS017+18	10-23D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	0,00	7,00	25,00
12	FS025+26	7-18D_A+B	1,25	1,25	6,25	5,00	2,50	0,50	1,25	0,00	7,00	25,00
13	FS032	-10-23D	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
14	FS033	-7-18D	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
15	FS019	-10-23DmLink	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
16	FS027	-7-18DmLink	1,25	1,25	0	0	2,5	0,5	1,25	0	18,25	25,00
17	FS017+18	-10-23D_A+B	1,25	1,25	6,25	0	2,5	0,5	1,25	0	12	25,00
18	FS025+26	-7-18D_A+B	1,25	1,25	6,25	0	2,5	0,5	1,25	0	12	25,00
19	Adenosine	Substrate only	1,25	0	0	5	2,5	0,5	1,25	3,75	10,75	25,00

 

 

#			Tris HCl ph 7.5	DNAzyme A	DNAzyme B	Substrate	NaCl	MgCl2	SDS	Kan	H2O	Final
20	FS032	10-23D	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
21	FS033	7-18D	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
22	FS034	Kan	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
23	FS035	Kan I	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
24	FS036	Kan II	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
25	FS037	Kan III	1,25	1,25	0	5	2,5	0,5	1,25	1,25	12	25
26	FS032	10-23D	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
27	FS033	7-18D	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
28	FS034	Kan	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
29	FS035	Kan I	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
30	FS036	Kan II	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
31	FS037	Kan III	1,25	1,25	0	5	2,5	0,5	1,25	0	13,25	25
32	Kan	Substrate only	1,25	0	0	5	2,5	0,5	1,25	1,25	13,25	25,00
33		Substrate only	1,25	0	0	5	2,5	0,5	1,25	0	14,5	25,00

 

Results and Outlook:

Positive controls worked, Adenosine dependency could be detected for one candidate.

▼2015-09-10 Click reaction of Label-, Label AU RNA [1, 2, 3]

For 51.5µL	Cstock	Cfinal	V[µL]
Phosphate Buffer - pH 7, 0.1M	100mM	50mM	25
Alexa 488 azide	10µM	400nM	2
RNA	1µM	200nM	10
CuSO4	20mM	1mM	2.5
THPTA	50mM	5mM	5
NaAsc	100mM	1mM	0.5
H2O			6.5

 

• Alexa 488 azide was solved in DMSO
• Incubation at 37 °C for 12-14 hours (overnight)

Beispieltitel 1

The use of restriction enzymes is no option for cloning if functional nucleic acids contain the recognition sites for enzymes used in standard restriction cloning, as described in RFC 10, or other suitable restriction enzymes. Although techniques using Type IIs restriction enzymes like Golden Gate assembly do not leave scars after cloning, a Type IIs recognition site within the functional RNA sequence may greatly reduce the efficiency and fidelity of a Golden Gate based cloning attempt. Therefore, the method of choice for reliable assembly of the sequences into a standardized vector has to be based on homology at the interfaces of the parts to be fused. As the DNA templates for functional RNA are commonly synthesized de novo, the addition of overhangs upstream and downstream the functional sequence does not appear to be challenging. Equally those extensions can be added during PCR with primer overhangs.

Beispielbild 1

Cooles neues Feature

Google hat neue Effekte

The term “functional RNA” covers a wide range of noncoding natural and synthetic RNA. These RNAs do not need to be translated into protein and are able to fulfil their function either by simple Watson-Crick basepairing interactions or by a more complex formation of secondary structures. Noteworthy classes of functional RNA relevant to the synthetic biologist include:

The term “functional RNA” covers a wide range of noncoding natural and synthetic RNA. These RNAs do not need to be translated into protein and are able to fulfil their function either by simple Watson-Crick basepairing interactions or by a more complex formation of secondary structures. Noteworthy classes of functional RNA relevant to the synthetic biologist include:

Beispielbild 1

Cooles neues Feature

Google hat neue Effekte

Beispielbild 1

Cooles neues Feature

Google hat neue Effekte

Comparison of the cost for the user of antibodies and aptabodies

Cost of antibodies for single use (smalles available size)

The prices of antibodies and aptabodies both in development as well as per use differs drastically. To get an accurate number of cost per use for antibodies, the prices of 4896 aptabodies offered by Thermo Fisher Scientific have been collected and plotted into a histogram. Thermo Fisher Scientific offers only one size for purchase, this equals then the price for the end user for single use. The aptabodies have been sampled pseudo-randomly from the Thermo Fisher Scientific database of "40,000+" antibodies, no currency conversion has been performed and shipping is not considered.

For comparison, the approximate calculated price of an aptabody is shown, see below how this cost is calculated.

Back to Introduction

Copper-catalyzed azide-alkyne cycloaddition (CuAAC)

Sharpless described the copper-catalyzed azide-alkyne cycloaddition (CuAAC) Kolb2001 also called click chemistry. This is due to the simple concept. It works under many different conditions with high yields and no byproducts. Mckay2014 A highly energetic azide reacts with an alkyne enabling a selective reaction that links these reactive groups to one another via a dipolar cycloaddition (Fig. 1). However this reaction requires a lot of activation energy.Zhang2005 Without catalyst the reaction is slow and results in a 1,4, 1,5 triazole regioisomer. To increase the reaction rate and to avoid this byproduct it is necessary to add copper as a catalyst. Copper (Cu(I)) proofed to be a suitable catalyst that rapidly yields a 1,2,3 triazole heterocycle.

Figure 1. Copper-catalyzed azide-alkyne cycloaddition (CuAAC)

Reaction scheme of the CuAAC. Adapted from: Source

Figure 2. Alkyne modification and click reaction of RNA

RNA was 3' modified with alkyne modified nucleotides using yeast Poly(A) Polymerase at 37 °C. Afterwards RNA was precipitated and used for copper click reaction with Alexa 488 azide.

The advantages of a click reaction are that it is very simple and works under many different conditions, as well as that the reaction results in high yields with no byproducts. The highly energetic azides react with alkynes enabling a selective reaction that links reactive groups to one another. To obtain the oxidation state of the copper sodium ascorbate is added to the reaction. Furthermore a ligand like THPTA is necessary to keep the Cu(I) stabilized in aqueous solution.

In order to use the above explained advantages of click chemistry for the labelling of DNA and RNA azide or alkyne modified nucleotides have to be incorporated into the sequence (Fig. 2). Martin et al. have shown that yeast Poly(A) Polymerase is able to incorporate modified nucleotides with small moieties to the 3’ terminus. To obtain an internal modification it is necessary to ligate two part of DNA or RNA to each other via splinted ligation. Winz2012

Summary

The specific monitoring of small molecules in biochemical reactions is problem that scientist tried to solve since many years. In this project we developed small molecule sensor (SMS) enabling us to analyze biochemical reactions in real-time using a fluorescent readout. Our implemented JAWS software generates us the best ATP-binding Aptamer which is fused to the Spinach Aptamer. In presence of ATP fluorescence will be emitted. This tool enables the sensing of small molecules like ATP that are part of biochemical reactions.

Major Achievements:

• Validation of the JAWS Software
• Sensing of ATP concentrations in biochemical reactions
• Establishment of a dual-fluorescent read-out system to sense small molecules in real-time
• Detailed monitoring of the ATP consumption as well as RNA strand synthesis

Abstract

The specific monitoring of small molecules in biochemical reactions is problem that scientist tried to solve since many years. In this project we developed small molecule sensor (SMS) enabling us to analyze biochemical reactions in real-time using a fluorescent readout. Using this innovative method, we were able to analyze classical in vitro transcriptions in detail by monitoring the ATP consumption as well as RNA strand synthesis simultaneously. Using a Spinach RNA Aptamer fused to an ATP-binding Aptamer RNA we can specifically sense ATP concentrations in real-time. Our implemented JAWS software generates us the best ATP-binding Aptamer. Thus we can even detect small changes in the concentration of ATP during RNA synthesis. To validate the JAWS software as well as to show the general feasibility of our fluorescent tool-box system we analyzed transcription efficiencies of different RNA polymerases, the influence of the buffer as well as the effect of inhibitors like heparin on transcription. Finally, the combination of the JAWS Software and the fluorescent readout enables the scientific community the possibility to target specifically any small molecule of interest in vivo and in vitro.