Design
QR Code Design
Set to focus the project on the topic of privacy and security we needed a tool that could contain a message that cannot be easily deciphered but has the capacity to store large amounts of information. To this end, we chose to use QR codes as they allow for storage of relatively large amounts of texts, while also offering the benefit of ease-of-use and error correction. This feature would allow the code to be read when not all bacteria grow as sucessfully
We have developed a software tool using Python and open source libraries to generate a secure QR code. Our design iteration consists of several steps:
- Encryption, in which the sensitive message is translated into an unreadable code that can only be accessed with the right password.
- Encoding, in which the encrypted message is translated into a QR code
- Decoding, a reverse step to translate back the QR code into encrypted message
- Decryption, using the right password, the encrypted message can be accessed back as a message
Iterative process of encryption and decryption of a message with QR code
Further information about our QR code design can be found on our
Software page.
The next challenge we faced was how to generate the physical QR code using biological material in the form of bacterial culture. During the course of our project, we explore 3 different methods to generate the physical QR code, each method comes with its own challenge in which we tried to solve.
- Etching (with laser machine)
In our earliest attempt to obtain a QR code, bacteria were grown on a plate, then we engraved the QR code pattern into the plate with a laser engraver. However, the drawback of this method is that we can only use one strain of bacteria, as the laser would burn away any empty spots with negative culture too. This would limit us to only one set of growing conditions, rather than the desired multi-value set. As such, the physical safety aspect is not sufficient to provide any benefits.
- Stamping
The next method we explored was to generate a physical stamp using a 3D printer, This stsamp was dipped into a bacterial culture, and then stamped on a plate. Initially, we manually create the 3D model from the 2D QR code image using CAD software. As this is a laborious process, we created a python script to automatically generate the 3D model. We also created different versions of the 3D stamp to increase the resolution of the QR code and to allow the use of more than 1 strain of bacteria in the QR code. Nevertheless, the stamping method was still considered arduous.
- Bio-ink printing
In our final approach, we combine the use of bioink and modified our 3D printer into a bio-printer. This method is superior to the other two as it can accomodate the use of more than one engineered strain. The QR code instantly forms as the bioink touches the plate and the bioink allows for induction as well.
To see the result of QR code from each method, refer to our
Results page.
Bioprinter and Bioink
Our hardware team has designed an in-house bioprinter by modifying a commercial Velleman Vertex K8400 printer. The commercial printer was meant for solid material, while we are working with liquid bio-ink. Thus, we added an external peristaltic pump to control the flow of the bioink and a tube to connect the bioink to a nozzle. For more precision printing, we used micropipette tips as nozzles. In addition, we also developed software to translate instructions for the printer to create a QR code (called gcode).
Additional information about the bioprinter is available on our
Hardware page.
The bio-ink was developed by mixing bacterial culture with sodium alginate. In conjunction, for printing we use media that has been supplemented with calcium chloride. Upon contact between sodium alginate and calcium chloride, calcium alginate hydrogel is formed and the ink instantly solidifies on the plate. This causes entrapment of bacteria inside the hydrogel into the plate. Moreover, the use bio-ink may allow for chemical induction by infusing the inducer into the bio-ink. We have thoroughly optimized formulations of the bioink and characterize the viability of two chassis organism,
E. coli and
V. natriegens inside the bioink.
Principle of bio-ink. Upon contact, sodium alginate and calcium chloride will form a hydrogel, entrapping bacteria inside the ink
To complement our bioink, we used strains of bacteria with GFP and RFP under the control of a tetracycline-induced promoter (pTet).
Further detail about our bioink formulations and characterization can be found on our
Results page.
Strain engineering
Throughout the project, we created several strains of bacteria. It is in our best interest to characterize and improve different promoters strength, as promoters are the key controllers of growth and no growth phenotype that we used as physical encryption with the biological QR code. In particular, we are interested to characterize and improve promoters for use in
V. natriegens as this organism is still emerging as a chassis and many genetic tools has not been characterized in this organism.
Characterization of promoters
We chose to characterize 2 different promoters in
V. natriegens, namely mannose inducible promoter (pMan: BBa_J100303) and arabinose inducible promoter (pBad: BBa_K808000). To measure their strength, we fused the two promoters with mCherry and later measured the strength of the fluorescence.
New promoter
We designed a new constitutive promoter for
V. natriegens (P1: BBa_K3171171) and for characterization we compared it with weak (BBa_J23106) and strong(BBa_J23102) promoter from the Anderson promoter library. All these promoters are fused to mCherry and then we measured the strength of each promoter by measuring the strength of the mCherry fluorescence.
Plasmid map of P1_mCherry for characterization
Improving the Promoter
We aim to improve the inducibility of two different promoters, pTet and pBad. To do so, we employ the synthetic promoter library (SPL) screening method from the iGEM Denmark 2010 team. In this method, we randomized the sequence of the promoter in between the -10 and -35 regions by using a randomized primer during PCR. The promoters obtained from the PCR were then fused to mCherry and the fluorescence intensity measured.
Plasmid map of SPL promoter screening for pTet
Plasmid map of SPL promoter screening for pBad
Characterization and improvements of our engineered strain can be found on the
Results page.
Biological containment (Auxotrophy)
We understand that safety is a critical issue and need to be addressed carefully in the project. One essential part of our safety aspect is to ensure containment of biological genetic material in case of accident and spill to the environment. We aim to create an auxotrophic strain which depend on the external supplementation of amino acid to the growth media. Thus, in case of spill, this bacteria strain will not be able to grow in the environment and will reduce the risk of genetic material transfer.
To achieve an auxotrophy strain, we target to knock out Histidinol dehydrogenase (HisD) gene which is important for histidine synthesis. We knock out the HisD gene with the use of CRISPR-Cas9 system. 3 pairs of gRNAs (BBa_K3171180, BBa_K3171181, BBa_K3171182) were designed to target the HisD gene. Targeted regions of the gRNA are shown below:
The gRNAs are then cloned into pJOE8999 plasmid downstream to a strong semisynthetic promoter (pVanP). The plasmid also bears sequence for Cas9 protein under the control of mannose inducible promoter (pMan), which is induced in the absence of glucose and the presence of mannose. In addition, the plasmid pUC origin of replication for
E. coli, temperature-sensitive origin of replication (pE194TS) for plasmid curing and a kanamycin resistance gene (kanR). The plasmid map can be found below:
Plasmid pJOE8999 as one-plasmid system for CRISPR-Cas9
In addition, since
V. natriegens does not have non-homologous end-joining (NHEJ) repair mechanism and instead has to use homologous directed repair (HDR), we also designed 3 different lengths of homologous sequence (BBa_K3171177, BBa_K3171178, BBa_K3171179) for repair templates.
The results of experiments with auxotrophic strains can be found on our Results page.
