iGEM Groningen 2019 :: Results

Results

At the start of the project we set out several goals so our eventual protocol would be easy to use, robust, reproducible and safe.

Goals:

The protocol and software accompanying the protocol should be easy to use.
The developed method should give consistent results.
Several strains have to be used in the same code for the code to be hard to crack.
During shipment, the code should not be visible, and only upon incubation under the right circumstances the code needs to becomes visible.

Achievements:

We created a protocol for precise bacterial disposition using 3D-printed stamps and a modified 3-D printer.
We have developed custom alginate based bio-ink that forms a hydrogel upon contact with a calcium coated surface.
By printing on a base without nutrients, we prevent the bacteria from leaving the hydrogel increasing the precision of our protocol.
We show that Escherichia coli and Vibrio natriegens stay viable inside the printed hydrogel when printed on an agar base without additional nutrients in several conditions.
We show that modified E. coli in the hydrogel are inducible with chemicals when stored at 4°C for several days.
Our software[link to software] is easy to use and by providing the program with the information you want to encrypt and a password, an AES encrypted QR-code will be generated in computer aided design (CAD) software.
This QR-code can then be converted to G-code which can be used by our modified 3D-printer.
Our in-house developed pattern recognition software is then able to scan the QR code using any camera. Upon supplementation of the password, the message will be revealed.

1. Bacterial Deposition

For consistency, we need to be able to deposit genetically engineered bacteria in the shape of a QR code with high precision. For this to become reality we explored several methods of pattern creation namely etching, stamping and printing.

1.1 Etching

After trying out several protocols and settings, we were eventually able to use a laser engraver to etch a QR code into a plate with bacteria. Here the bacteria are spread over the surface of the plate before being treated with a laser. After incubation overnight, the pattern appears (Figure 1). This method produces readable QR codes.

Etched Plate — Figure 1 - Etched QR codes in a plate with *E.coli*, the pink colour comes from J0445.

However, anyone that gets these plates would be able to scan the code. For our QR code to be secure, several strains need to be used that become visible or grow under certain conditions. This is not employable using this method. In any case, we show that a laser can be used to kill bacteria with high precision.

1.2. Stamping

To further test methods for precise bacterial deposition, stamps were explored. The stamps were rested in a solution with bacteria before tapping it gently on an agar plate. The first generation of 3D printed stamps were solid (Figure 2).

Ultimately this resulted in blurry codes (Figure 3). The next generations of 3D printed stamps therefore were pixelated, allowing excess fluid to flow away in the grooves eventually resulting in a clearer, readable QR code (figure 4).

Additionally, we tried out prongs to be able to correctly orientate several stamps, using several strains of bacteria (figure 2). This is needed to increase the security of our procedure. However, we ran into problems with manually aligning the stamps regardless of the prongs. Printing one stamp takes about 3 hours. This makes the whole procedure quite time-consuming and far from easy to use, especially in the case of multiple stamps per code. These issues prompted us to further develop other methods in our pipeline, but not before we were able to scan a code using our image processing software [link to software] (figure 4).

Black and White QR — Figure 4 – Stamped plate, processed with our software.

1.3. Printing of bio-ink using a 3D printer

1.3.1. Conversion of a 3D-printer to a bioprinter

In order to print a biological QR code, we set out to modify a commercially available 3D printer (Vertex K8400, into a bioprinter (link to hardware). First the filament extruder head, the part that pushes the filament to nozzle, is replaced with a peristaltic pump head (figure 4). A pipette tip (10 µL tip) connected to silicon tubing (inner diameter of 1.6 mm) was mounted on top of the print head (figure 5). For more than one type of bio-ink, we can potentially use the second extruder of the printer and modify it similarly. The silicon tubing is plugged in the reservoir via the peristaltic pump head, setting the printer up for handling liquid biological ink (bio-ink). This bio-ink solidifies within seconds upon contact with the printing surface (link to bio-ink on this page). The instructions (G-code) responsible for instructing the printer to create a QR code can be created using our custom software (link to software). We can control the stepper motor driving the peristaltic pump head to control and adjust the flow rate of bio-ink during printing jobs. The temperature requirements for printing are removed from the G-code to allow the printer to print without heating the nozzle. These adaptations effectively set up our printer for printing multiple engineered bacteria using bio-ink.

1.3.2. Resolution and reproducibility of the printer.

Our printer needs to be able to consistently print squares, since they are in every QR code. The consistency of the printer was determined by printing a square inside a square in a petri-dish using the developed bio-ink [link to bio-ink]. Food dye was added to the ink to ensure visibility. Without changing any parameters in the G-code, we printed four plates consecutively as shown in figure 6. What can be seen is that at the beginning of a line, more ink is deposited on the plate. This effect is increased due to the beginning and end of these lines coinciding, creating a blob (figure 6). Provided these discrepancies are not to big, we believe that this would not prevent a QR code from being readable. We demonstrate that our printer can print squares with proper consistency needed for our projects aim.

To characterize the spatial resolution of printed structures, 2 circles and a square were printed to evaluate the divergence in the same print and evaluate the resolution. The line width was measured using ImageJ Fiji software package (imageJ) and is shown in figure 7. The line width generated by our printer varies. At the beginning the line is thicker due to the blob. Ultimately we were able to obtain line widths as low as 0.5 mm. Optimization of the extrusion parameter is potentially a way to decrease the variation in the line width. However, having a consistent line width is not necessarily required for the end goal of our project.

Because we have control of the flow, we are able to print dots. The liquid droplets can spread. When dots are printed to close to each other they can merge (figure 7). We found that printing within 2 mm of each other can sometimes allow them to merge, since alginate cross-linking is not instantaneous. Potentially this can be prevented when increasing the time between dots that are close to each other. We used our software (link to software) to convert the word “QRoningen’’ into a QR-code and transform the code to G-code instructions for the printer. After several tries, and modifying the extrusion parameters in the G-code, we were able to print a QR-code.

1.3.3. Bio-ink

Bio-ink was developed to allow for printing using mild conditions suitable for live bacteria. Sodium alginate inside the ink supplemented with nutrients is in liquid form when moving through the tubing but solidifies upon contact with calcium ions in the printing surface. The calcium ions cross-link the alginate molecules forming a stable gel within seconds [2]. Several concentrations of alginate (1 % w/v to 10 % w/v) in the ink and CaCl2 (0.05 – 5 M) in the printing surface were varied to find optimal values [1]. We also found that low concentrations of alginate would result in poor gel quality (below 1.5 %) and high concentrations of alginate resulted in viscous ink that would limit control of the flow or was impossible to move. Very high concentrations of CaCl2 prevented agar from becoming solid (over 1M) and would increase the speed of solidification in addition to being undesirable for bacteria due to osmotic stress. Furthermore, these high concentrations would allow for the ink to solidify in the tip, preventing extrusion and decreasing the robustness of the procedure. At concentrations as low as 0.05 M CaCl2 we found that the speed of solidification was still within seconds when using small volumes. Hence, we use 0.05 M CaCl2 in the printing surface and 2 % w/v sodium alginate in the ink in our printing procedures.

1.3.4. Viability of E. coli and V. natriegens in printed bio-ink.

The bacteria inside need to be able to survive when incorporated in the printed ink. To evaluate this, E. coli and V. natriegens were printed on agar with nutrients using our custom ink to evaluate the survivability. After storing unprinted ink and plates with the printed bacteria from 6 to 72 hours at room temperature, samples were treated with a sodium citrate solution to resolubilize the gels. The amount of bacteria inside the samples were evaluated using the plate count method (Figure 9). Interestingly, we found that E. coli exhibits almost no growth when inside the unprinted gels. Moreover, V natriegens seems to become less viable over time when stored inside bacterial ink. The hindered growth of both organisms could be explained by the absence of shaking of the tubes, reducing aeration. These findings suggest it is best to use freshly prepared ink for printing. When printed on a supporting structure that has extra nutrients, bacterial growth is observed in both organisms (Figure 9). A ring of bacteria was observed around the gels. Bacteria were also observed on top of the printed gels. The nutrients inside the agar potentially allow for the bacteria to grow and move outside of the bounds of the gel and grow further. This overgrowth of bacteria is unwanted in the case of QR-code printing, since it could disturb the readability of the code.

By supplying no additional nutrients in the plate we aimed at confining the bacteria to their initial printed position. The plates with the printed ink were kept for varying amounts of time ranging from 0 h to up to one week at room temperature before a viability assay was performed (Figure 10). In the first 24 hours growth is observed in both organisms. Thereafter no growth was observed. These findings are in line with previous work [1], and suggest that the organisms keep growing until a cap is achieved, after which the limited amount of space and nutrients, provided in the ink limits growth. Indeed, here we show that the printed gels are capable of supporting growth of bacteria, without additional nutrients.

For the purpose of our project it is beneficial that the code is not visible until induced. We anticipated that by storing the plates with printed ink in the fridge at 4°C for 3 days, we could halt bacterial growth until growth is needed. Another viability assay was performed to confirm this (figure 11). Note that there are no nutrients inside the plate and that the vibrio culture was grown for only 6 hours prior to be made into ink. The viability of E. coli dropped 3 fold and the viability of V. natriegens dropped 4 fold when stored at 4°C for 3 days inside printed ink. This fold change is bigger than is observed in liquid culture in literature when V. natriegens is kept at 4°C [3]. Potentially this is caused by the limited amount of nutrients. Here we show that the bacteria stay viable when stored at at 4°C, for example during transport. This can be used to prevent the code from appearing during transport.

1.3.5. Inducibility inside the printed ink.

1.3.5.1. Bio-film induction

Qroningen hinges on inducibility of constructs inside genetically engineered bacteria inside our printed alginate gels. Dr Srikkanth Balasubramanian (link to attributions) was kind enough to send us two plasmids used to show ability of biofilm formation inside the physical support of printed alginate hydrogels [4]. One plasmid encoding for pSB1C3-GFP-pRha-CsgA allowing for rhamnose inducible CsgA production accompanied by constitutive GFP production. The other plasmid encoded for pSB1C3-GFP offering only constitutive GFP production. Bacterial ink harboring these plasmids were deposited on print plates with rhamnose (0.5 %). The experiment was performed on plates with inducer and plates without inducer. After induction, the plates were treated by adding a 0.1 M solution of sodium citrate and incubating at room temperature for 3 hours at 70 rpm. The liquid was removed after 3 hours effectively removing the printed gels from the plates. After treatment, the plates were observed using a camera and the Typhoon FLA 9500 biomolecular imager (figure 12 and 13). Biofilm formation can be observed on plates that have the plasmid and the inducer after 2 days. Biofilm manifests itself as a thin film where the alginate gel used to be. On plates without additional nutrients no biofilm formation is observed after 6 days

The induced CsgA protein subunits are transported outside of the bacteria where they self-assemble into CsgB proteins that form curli amyloid fibers [5]. These form the protein core of the extracellular biofilm native to E. coli [4–6]. These findings suggest that a considerable amount of nutrients is needed for formation of such a biofilm. More importantly, these findings show that substances inside the alginate gel can diffuse from the printing substrate into the alginate gel and exert an effect on the resident bacteria.

1.3.5.2. GFP and RFP fluorescence when inducers are supplemented to the plate at a later time point.

In the previous experiment, the inducer was in the supporting structure. For our application it would be ideal if inducers could be added later. This was evaluated by depositing E. coli harboring a tetracycline inducible green fluorescent protein GFP on print plates with nutrients [link to sonja, attributions]. A certain amount of ink was deposited on the plate. Later, an equivalent of liquid containing commonly used concentrations of anhydrous tetracycline (aTC) and tetracycline (TC) were added to the plate. After 24 hours of incubation, the plates were observed in the typhoon FLA 9500 biomolecular imager and the fluorescence was quantified using imageJ. The area of interest was selected and measured in triplicate for each continuous area of cells on each sample. The results of which are shown in figure 14. This indicates that inducers can be added later to the plate and are able to diffuse through the printed ink.

For our application, it would be ideal to send a plate in refrigerated conditions, add an inducer and then observe the plate. In this experiment, E. coli containing a tetracycline inducible red fluorescent protein (RFP) were printed using a pipette on a printing plate containing no additional nutrition in triplicate [link to sonja, attributions]. A less-toxic tetracycline derivative aTC, was added 48 hours after storage at 4°C as inducer. The plates were incubated and imaged 24 hours later using the typhoon FLA 9500 biomolecular imager. The fluorescence was quantified with imageJ. Fluorescence intensity was measured by measuring the average intensity of a fixed area with bio-ink in triplicate (Figure 15). What can be seen is that the intensity of the plates that have the construct are higher, this is probably due to leakiness of the promoter. Furthermore, when 1 µg/mL was used, a significant increase in fluorescence can be observed thus indicating our proposed method is viable. Note that the amount of inducer used here to elicit a response was higher than in the previous experiment. This can be explained because bacteria become less responsive over time. We would have to carefully evaluate inducer concentrations of all of the constructs we use. Non-toxic inducers are preferred for this reason.

Overall these experiments show that our concept is achievable. We demonstrate that we can deposit bacteria using a modified 3D-printer as dots instead and squares. This gives us spatial control over bacterial deposition. A feat that, to the best of our knowledge, has not yet been achieved before using a modified commercial 3D printer. We have also shown that bacteria are able to survive inside the ink in refrigerated conditions, staying responsive to inducers. Inducers are able to diffuse through the printed gel and can therefore be added later to an already printed plate. Not using nutrients in the ink allows us to prevent bacteria from leaving their initial place on the plate preserving the resolution of the code over time. In multiple experiments using diverging constructs we show that genetically engineered E. coli cells are inducible inside our printed bio-ink.

2. Genetic engineering and characterization

2.1. Characterization of promoters and constructs

The characterization of our constructs focuses on inducible promoters. These regulatory parts are of utmost importance to our project as they allow for environmentally controlled phenotypes. Only if the correct key is added - in this case inducers - the QR code will be readable.

2.1.1. Constructs in V. natriegens

Because of its astonishing growth rate of only 7 minutes, V. natriegens is ideal for our purposes. The quicker growth means a quicker readout of the message. A lot of procedures are not optimized yet for use in V. natriegens though, so we decided to use our project to also help with the popularization of this intriguing organism and provide a step forward in exploring all its possibilities. We characterized the inducible promoters using mCherry as reporter as we were advised by V. natriegens expert Tanya Tschirhart.

2.1.1.1. P1 Promoter

We contributed a new basic part, the native P1 promoter from V. natriegens (BBa_K3171171). The gene under the control of this promoter is transcribed constitutively. Fluorescence measurements of the construct in E. coli and V. natriegens reveal a constitutive expression of the reporter mCherry for both organisms. Interestingly our measurements show that the expression levels in E. coli are a lot higher than in V. natriegens as the fold change is 225 for E. coli compared to 3 for V. natriegens.

2.1.1.2. pBAD in V. natriegens

The pBAD promoter in part BBa_K808000 is one of the most commonly used parts in iGEM and synthetic biology. We characterized its use and inducibility in V. natriegens. Our measurements show very high expression levels for a concentration of 0.5 % (w/v) arabinose and a very low basal level of expression without the inducer and the fold change amounts to 1127. In conclusion, pBAD offers a tight and regulation of expression in V. natriegens.

2.1.1.3. pMAN in V. natriegens

The mannose inducible promoter is part of the pJOE8889 plasmid and controls the expression of the Cas9 enzyme. Since we designed all CRISPR components to work with this plasmid we also had to make sure that Cas9 would be expressed upon addition of mannose. There are no reports in the literature on the use of this promoter so we characterized it with our reporter mCherry. Moreover, V. natriegens cannot utilize mannose as a carbon source. Addition of 1 (w/v) % led to a 2.5 fold increase of expression within 12 hours.

2.1.2 Constructs in E. coli

We characterized the pBad (BBa_K808000), pTet (BBa_R0040) and pMan (BBa_J100303) promoter using mCherry as a reporter. For pBad we observe a 10 fold increase of mCherry expression incubating them for 8 hours with 1 % arabinose (figure 19). Induction with anhydrotetracycline for 8 hours let to a 20 times increase of mCherry fluorescence (figure 20). We also characterized the mannose inducible promoter which was constitutive in our measurements (figure 21).

2.2 Synthetic promoter libraries

We engineered two synthetic promoter libraries with the aim to improve inducibility and function in E. coli and V. natriegens. We selected the arabinose (BBa_K808000) and tetracycline (BBa_R0040) as targets for our design following the recommendation from Tanya Tschirhart, who experienced a lower induction of both promoters in V. natriegens, compared to E. coli. In order to construct the library we followed the design from Denmark iGEM team 2010. We designed primers that would amplify the plasmid with the original pTET_mCherry or pBAD_mCherry construct and yield randomized sequences between the -35 and -10 region of the promoters. Additionally, the overlapping part of the promoter contains a restriction site that was used for circularization of the plasmid.

The constructed plasmids were transformed into both E. coli and V. natriegens and appearing colonies were screened for inducible fluorescence with the corresponding inducer. In total 271 clones were measured with and without inducer, 245 for E. coli and 26 for V. natriegens. Numbers for V. natriegens are lower because of low transformation efficiency and the data are not shown. Data for all screened E. coli is displayed in the heat map, the first third of table representing data from pBAD, the rest being pTET. The presented numbers represent the fold change, that was achieved by dividing fluorescence in the presence of inducer by the fluorescence without it.

This data allowed for the selection of 10 most promising clones. Again, fluorescence upon induction was measured, this time in duplicate. Most did not show an increase in fluorescence with the addition of the inducer. However, clone 6 from the batch induced with anhydrotetracycline reached a 34 times higher fluorescence in the presence of the inducer.

Apart from this strong inducibility we also checked the basal level of fluorescence. Comparison with the background fluorescence of E. coli reveals that indeed there is no leaky transcription of mCherry in absence of the inducer.

We finally isolated and sequenced the plasmid to get the sequence of our optimized tetracycline inducible promoter.

2.3. CRISPR

Knock out using CRISPR applies the Cas9 enzyme, which with the help of a gRNA finds a target and introduces a double stranded break. If such a break happens within the genome of an organism, it has to be repaired to stay viable. One method to repair the break is using a homologous part as a template to ensure functionality of the repaired sequence. In genetic engineering, we customize both the gRNA, i.e. the breaking point, and the homologous part, i.e. the final sequence after the repair.

Our motivation to apply CRISPR was the knock out of the HisD enzyme, which is responsible for the last step of the histidine biosynthesis pathway. Missing functionality of this enzyme would lead to auxotrophy. Using a histidine auxotroph would require the addition of this amino acid if you want to read our message, adding another layer of security.

We designed three gRNA target sites using Synthego software and three homologous parts of different length complementary to the up and down stream region of HisD. A summary of all these parts can be found in our basic parts page. We cloned the gRNA into the pJOE8889 plasmid and transformed them along with the homologous parts. The transformations were first plated on medium plates containing the resistance marker kanamycin and the inducer mannose. Colonies that appeared within 2 days were plated on minimal medium with or without histidine to identify auxotrophy. Clones that would grow only if histidine is supplied are auxotrophic. However, this screening revealed that no auxotrophy was achieved so the protocol should be revised and optimized in the future.

Literature References:

[1] Lehner, B.A.E.; Schmieden, D.T.; Meyer, A.S. A Straightforward Approach for 3D Bacterial Printing. ACS Synth Biol. doi: 10.1021/acssynbio.6b00395
[2] Kuo, C.K.; Ma, P.X. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: Part 1. Structure, gelation rate and mechanical properties. Biomaterials. doi: 10.1016/S0142-9612(00)00201-5
[3] Weinstock, M.T.; Hesek, E.D.; Wilson, C.M.; Gibson, D.G. Vibrio natriegens as a fast-growing host for molecular biology. Nat Methods. doi: 10.1038/nmeth.3970
[4] Hammar, M.; Bian, Z.; Normark, S. Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli. Proc Natl Acad Sci U S A. doi: 10.1073/pnas.93.13.6562
[5] Wang, X.; Smith, D.R.; Jones, J.W.; Chapman, M.R. In vitro polymerization of a functional Escherichia coli amyloid protein. J Biol Chem. doi: 10.1074/jbc.M609228200
[6] Spiesz, E.M.; Yu, K.; Lehner, B.A.E.; Schmieden, D.T.; Aubin-Tam, M.E.; Meyer, A.S. Three-dimensional Patterning of Engineered Biofilms with a Do-it-yourself Bioprinter. J Vis Exp. doi: 10.3791/59477

Team:Groningen/Results