As a communication system, the use of CryptoGErM requires collaboration and so, to test our system, we asked the other iGEM teams in the Netherlands to help us with this. However, we also collaborated with iGEM teams abroad. This article describes all our collaborations with the other iGEM teams.
Sending our message in spores to iGEM Wageningen and Eindhoven:
The idea of CryptoGErM is to send information integrated in the sequence of DNA. Our system is using highly resistant spores in which the DNA is stored. Spores are then transmitted to the receiver who can get the DNA from them and therefore the stored information as well. We wanted to prove that this form of information transmission works. Therefore we decided to cooperate with two other iGEM teams from the Netherlands and send our CryptoGErM spores with a secret message to Wageningen and Eindhoven. Read the accompanying letter here. Follow this link to see how we constructed the message B. subtilis strain.
We went through all the 6 steps of our project. The message was encrypted with our program and converted to a DNA sequence. This sequence was synthesized by IDT. When this synthesized DNA sequence was obtained, we integrated the message sequence into the genome of Bacillus subtilis. Subsequently we sporulated B. subtilis and spores were obtained. These spores together with primers were sent to Wageningen and Eindhoven via mail under GMO conditions. Spores and primers were in the mail from the 6th September till the 9th. In this case we sent primers for PCR amplification of the message because we assumed the other teams did not include a whole genome sequencing in their finance plan. We sent this CryptoGErM protocol along with the message to the mentioned teams. The short version is, they germinate the spores on spectinomycin LB agar plates and from those colonies the message can be acquired via colony PCR. Primers for the PCR are provided (primer sequences can be found here). This PCR product is sent for sequencing. The received sequence can be copy pasted to our decoding program. Now they only need to know the digital key that we used to encrypt the message. We were kind enough to tell them the digital key beforehand.
It works! The iGEM team Eindhoven and Wageningen could read our message which was: “The world is full of obvious things which nobody by any chance ever observes.” The digital key to decrypt this message was: ”Autoclave after reading!” We hope the teams did this with our message after the decoding.
Characterization of the BioBricks (BBa_K1913011) B12 biosensor and (BBa_K1913012) guanine biosensor from the iGEM team Wageningen 2016
The aim of the Wageningen iGEM 2016 project is to construct an Escherichia coli strain that effectively kills the mite Varroa destructor within a bee hive. One step towards this goal is the design of a system that regulates gene expression upon presence of the V. destructor mite, which is indicated with an increase of vitamin B12 and guanine concentration. Vitamin B12 and guanine riboswitches are used to design two constructs that provides E. coli with a plasmid that regulates gene expression based on the vitamin B12 or guanine concentration. For the design, the sequence of the btuB gene vitamin B12 riboswitch of E. coli and the sequence of the xpt-buX operon guanine riboswitch were used. The sequences have been elongated with fifty amino acids downstream of the riboswitch in the original genomic sequence of B. subtilis and E. coli to potentially increase the stability. It is followed by the sequence of the TetR system: a quad-part inverter based on tetracycline that inverts the signal since both riboswitches stop gene expression upon binding their respective metabolite. The TetR QPI inverter reverses this process and cause protein expression when the riboswitches have bound the ligand of interest. This is the desired outcome in the overarching project: a V. destructor sensing bacteria would ideally start with the production of a toxin in presence of the mite. Instead of a toxin, mRFP is used as a reporter gene.
iGEM Wageningen 2016 sent us these two BioBricks (BBa_K1913011 and BBa_K1913012) they constructed, one to detect guanine and one for vitamin B12. We helped them to show that the RFP expression is controlled by the concentration of either vitamin B12 or guanine. We could show an increasing RFP signal with increasing concentration of vitamin B12 and guanine using a photometric measurement with the plate reader (see protocol below). We also took pictures of the cells at the microscope with and without vitamin B12 or guanine.
1. Photometric measurement of the OD600 and mRFP fluorescence
Grow E. coli in 3 ml LB with 35 μl/ml chloramphenicol at 37°C and 220 rpm for 16 h. Prepare a transparent 96-well plate with a total volume of 250 μl. Provide 175 μl LB and 25 μl of the appropriate concentration of guanine or vitamin B12. Dilute cells to an OD600 of 0.5 with LB and add 50 μl of this dilutions to each well. To measure the fluorescence the excitation and emission of mRFP was set on 584 nm and 607 nm, respectively, with a bandwidth of 5 nm. The absorbance was measured at 600 nm. Measurements were taken every 20 min at 37°C with the plate reader (Varioskan LUX Reader, Thermo Fischer). The results can be seen in Figure 5 and 6.
The Figure 4 shows that E. coli expresses RFP in response to the concentration of vitamin B12 in the medium.
Figure 5 does not show that the expression of RFP in E. coli with the biosensor for guanine is actually correlating with its concentration.
The difference in RFP expression in response to the concentration could even be seen by eye. See Figure 6 and a photo of the 96-well plate used for the plate reader experiment.
2. Microscopy of the E. coli with the biosensor
Grow E. coli in 3 ml LB with 35 μl/ml chloramphenicol at 37°C and 220 rpm for 16 h. The control grows in LB only. The sample is grown with 2 mg/L vitamin B12 or 0,1 mg/L guanine. 1,5 μl of cell culture is added to a microscopy slide covered with a thin layer of 1,5 % agarose (see microscopy protocol). Microscopy pictures were obtained with objective: Olympus 100X/1.40, camera: CoolSNAP_HQ / HQ2-ICX285 and software: Resolve3D softWoRx-Acquire Version was used. Phase contrast: Filter POL 50% 0,8 s exposure and red fluorescence: mCherry 32%, 0,8 s exposure was applied.
Vitamin B12 sensing BioBrick BBa_K1913011:
Guanine sensing BioBrick BBa_K1913012:
Both experiments show that the RFP is already expressed in regular LB. This is probably due to traces of guanine and vitamin B12 in LB medium. Nevertheless the pictures also show that the RFP signal increases with the addition of guanine and vitamin B12.
Dutch meet up with iGEM Wageningen, Eindhoven, Leiden:
At this meeting we shared our first presentations and gave each other feedback. It was very useful for starting up our iGEM projects. Social snack was included. Special thanks to Wageningen for hosting us!
Team Göttingen is aiming for the optimizing of the biotechnological production of vitamin B12. They sent us a survey with an interest to know what we know about the 'red gold'. Furthermore they wanted to evaluate the general understanding of biotechnology and which chances we see in it. This survey contained questions for example: „Are you aware of the physiological functions of vitamin B12 in the human body?“, „What do you think, in which of the following processes is vitamin B12 involved?“ and „Are you aware of the biochemical functions of Vitamin B12 in any living cell?“.
iGEM Paris Saclay:
We did their survey and earned this badge. The survey was about Responsible Research and Innovation: This is an initiative from the European Commission to bring society and science together. It means that the scientists must work during the whole research process in order to make sure that they fulfill the values, needs and expectations of the European society.
iGEM METU HS Ankara:
The iGEM Team Metu HS Ankara 2016 contacted us about translation iGEM Protocols (Protocols like transformation or making competent cells) to our own language. They wanted to create a database consisting of protocols in different languages.
iGEM Tel Hai:
iGEM Tel Hai team 2016 is working on a new cure for Cystic Fibrosis, and as part of their project, they wanted to collaborate with the Cystic Fibrosis Foundation and with the Cystic Fibrosis Foundation of Israel. They decided to run a campaign called 65 roses, of which people being photographed holding a rose in order to raise awareness among the general public and raise money for those great foundations, just like with the ALS ice bucket challenge.
This campaign is worldwide. It is supporting the story of a girl diagnosed with CF, who thought for the first time she heard about the diagnosis that she has 65 roses. Therefore we grew a rose for her in our lab.
Nowadays, digital espionage is becoming a problem. Its main goal is to steal information of private parties and use it in an illegal way. Our project consists of the development of a system to store encrypted information in bacteria. In addition, the information will be secured with several biological layers that make it digitally uncrackable. We would really like to know how society sees our problem, therefore we made a survey. This survey contains questions like; “Would you send an important highly-secret letter to someone you trust by post or email?“, „Would you trust bacteria to store your highly-secret information?“. More about this survey? See this link.
This survey was sent to most of the participating iGEM Teams. The teams who participated are: University of Westminster iGEM 2016, Paris-Saclay iGEM 2016, UPO-Sevilla iGEM 2016, Paris Bettencourt iGEM 2016, iGEM Pasteur 2016, iGEM Sheffield 2016, iGEM Leiden 2016 and TU Delft iGEM 2016. In exchange the teams got our collaboration badge.
Very special thanks to iGEM XMU-China for the translation of our survey into Chinese.