Overview of constructed BioBricks
The BioBricks that were successfully constructed during our project:
Achievements
At the Jamboree we won the track award: Best Information Processing Project, gold medal and were nominated for:
- Best Software Tool
- Best Education & Public Engagement
- Best Wiki
View our poster, presentation slides or other results.
Medals
- Register and attend
- Deliverables
- Attributions
- Parts/Contributions
- Validated part/ contribution: BBa_K1930000, BBa_K1930001, BBa_K1930004
- Collaborations
- Human Practices
- Integrated Human Practices
- Characterization of a previous part: BBa_K823023
- Proof of concept
- Demonstrate your work!
Integration and recovery of messages in DNA
We integrated an encrypted message and a key sequence into the amyE locus of the genome of two different Bacillus subtilis strains. Both Bacillus strains were treated to form spores. Subsequently the spores were allowed to germinate, the DNA was isolated and the key and message sequences were amplified using corresponding primers. The amplified DNA was sequenced and we inserted the sequencing results into our encryption and decryption machine (link). We were able to recover our key and with the key we could decrypt the sequencing results for the message. We could recover our message stored in DNA! To test out the system under real life conditions we sent the message-spores and the corresponding primers to two iGEM teams in the Netherlands. They also recieved the key from us in order to encrypt the message. The iGEM teams from Eindhoven and Wageningen were both able to decrypt our message. Our concept is working!
CRISPR/Cas9 key deletion
We successfully annealed the sgRNA of the message and the key. The ligation of these with the vector pJOE8999 failed. We managed to amplify the two PAM sequences of 700 bp of the B. subtilis genome. We attempted to amplify the tetR from its plasmid backbone, unfortunately the PCR failed. This subproject should be repeated from the beginning.
Decoy experiment
In order to test the efficiency of selecting the correct key spores from decoy we conducted flow cytometer measurements. Additionally we observed the selection efficiency with a microscope. For this experiments we used the BioBrick BBa_K1930006 sfGFP in pSB1C3. It could clearly be observed that the antibiotic selection was successfull. Though, the expected outgrowing of the decoy cells in order to hide the key-cells could not be observed. In the future this experiment should be repeated to find a concentration of decoy cells that outgrows the key cells. Also the experiment should be repeated while using spores instead of living cells to adjust the experiment to real life conditions.
MIC and MBC of ciprofloxacin.
The MIC and MBC of ciprofloxacin on wild-type Bacillus subtilis 168 were determined to be between 160-170 nM (MIC) and 180 nM (MBC). Subsequently, the MIC of E. coli top 10 and B. subtilis 168 carrying the qnrS1 ciprofloxacin resistance gene was determined to be between 1000 - 2000 nM, and 400 - 500 nM, respectively. This is a significant improvement in antibiotic tolerance when compared to the MIC values of the wild-type strains (100-130 nM for E. coli and 170-180 nM for B. subtilis). Finally, the MIC value of a B. subtilis 168 isolate resistant to ciprofloxacin (obtained via directed evolution) was determined to be greater than 20,000 nM, which is a huge improvement in ciprofloxacin resistance over both the wild-type strain, and the strain carrying qnrS1. In the future, the qnrS1 and aac(6')-Ib-cr quinolone resistance genes could be introduced into this highly resistant strain for even greater ciprofloxacin resistance.
Molecular Dynamics modelling
We used molecular dynamics methods, in particular umbrella sampling, to identify a priori which strains of B. subtilis would be more susceptible to spirofloxacin. Thus, when the antibiotic was in its active state it would have a higher wild-type vs mutant killing ratio. However, due to the lack of consistent results in its experimental phase we decided not to continue with the photoswitchable antibiotic approach.
Characterization of the BioBrick BBa_K823023
We managed to successfully calculate the transformation efficiency of BBa_K823023. Transformation with 10 ng and with 100 ng DNA worked well for integration. BBa_K823023 could therefore be used as integration vector for the message and key in the genome of B. subtilis.
NucA kill switch – improvement of a part
In our project we constructed a system which will destroy the key DNA sequence if tetracycline or tetracycline analog aTc (anhydrotetracycline) is not be added while culturing the cells. For this construct four parts were needed. The main part was nuclease from Staphylococcus aureus (nucA), this was ordered from iGEM headquarters as an available BioBrick BBa_K729004 from iGEM team University College London 2012. We have tried to assemble our desired construct (see first step RBS+nucA), but after sequencing of this plasmid (RBS+nucA), we realized that the nuclease BioBrick (BBa_K729004) has an incorrect prefix. Due to time limitation, it was decided that we will not continue with cloning of our desired nuclease construct, but we will make an improvement of this part and we will try to fix the incorrect prefix.
Encryption and Decryption machine
The encryption and decryption machine can convert data into strings of DNA. Furthermore it can encrypt and decrypt the data using the Rijndael algorithm. It can also consider forbidden sequences such as restriction enzyme cutting sites. For now, extremely long datasets take up significantly longer. Further code optimization can fix this inconvenience.
Belief-desire-intention model (BDI)
The BDI model simulates the behavior of the potential users of CryptoGErM. Computational Agents are programmed according to an Artificial Intelligence (A.I.) architecture and placed into a virtual scenario where they compete for saving and sending a top secret message. By observing their A.I. behavior we understood how to make CryptoGErM as safe and effective as possible. We obtained a concrete proof that creating the whole system around multiple security layers was the best strategy to follow.
Random mutations modelling
This model explores the role of random mutations in the Bacillus subtilis bacteria. Considering how the encrypting and storing works, we have created a software which is able to virtually replace parts of the bacteria's DNA and check if the stored data would have been affected by a mutation. Out of 1000 simulations this happened only 3 times, so we deduced that our system is 99.97% accurate on a 556 base pair message. The software is also able to compute the maximum amount of base pairs that can be modified in the bacteria which corresponds to 400,320 bps.
Collaborations
We collaborate with teams from all over the world. We send our message and key to the teams Wageningen and Eindhoven, we analyzed a BioBrick for Wageningen and we filled in a few surveys. In addition we also send a survey to a lot of teams, which has also been translated to Chinese, thanks to XMU-China. Finally, we helped translating protocols to Dutch for METU HS Ankara.
Education
After a survey we found that 26% of the respondents did not know what bacteria are. We set it as our aim to enhance public awareness of bacteria and of genetic engineering to overcome this problem. Therefore, we invited more than 150 students into our lab to do some Bio-Art with colourful bacteria and gave a lecture about CryptoGErM. In addition, we guided/supervised several students in their high school thesis. They did their first cloning with GFP in E. coli and B. subtilis and experienced pros and cons of working with the different bacteria. After the Giant Jamboree we will be giving lectures about our project during a science event in Groningen.
Outreach
To communicate our project to general society we gave an interview on the local radio, engaged with them at the night of art and science, had a feature in the university paper and in the magazine of the biology study association. We had more visual representation through a videoclip made by Unifocus and even reached the national news agency (NOS). More in-depth communication of our project to the scientific society was conducted at a barbecue organised for the staff of the university, workshops at the Utrecht campus party and a presentation during the Dutch Biotechnology Conference and at the Groninger Biomolecular Sciences and Biotechnology Institute (GBB).
Having a fun summer
