Demonstrate
In order to realize our vision of a biological QR code we had to work in several fields and
optimize multiple aspects like: achieving the correct and precise bacterial pattern, strain
engineering and software related to encryption and printing of QR codes (Figure 1).
Figure 1 - Graphical depiction of our project.
Bacterial QR code
Laser Engraving
We show that for a single bacterial strain, it is efficient to laser etch the QR code into the plate
and achieve a clear and scannable result (figure 2). We seed a clear agar plate with a lawn of
bacteria and etch the plate under a laser CNC. The laser module we use has power of 500 mW and
wavelength of 400 - 460 nm. The software we use is Laser GRBL which is open source and requires only
a vector image of the encrypted QR code generated by our encryption software.
Stamping
In order to increase security, multiple strains, that are influenced by different inducing agents
have to be plated simultaneously. We show that the use of 3D printed stamps gives a readable QR code
(Figure 3). This stamp is used to transfer the liquid culture as ink onto an agar plate. Multiple
strains can be applied using half stamps that are aligned using protrusions on each corner. Further
optimization and automation of the stamping procedure can potentially allow for correct application
of multiple different strains.
Figure 3 - Stamps and the results of stamping.
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. We optimized our ink and
found that using 2% sodium alginate in the ink and 0.05 M calcium chloride in the printing
surface
is optimal (As shown in Results). We make the ink by growing the bacteria overnight and spinning
them
down. Then, the bacteria are resuspended in residual liquid (about 0.5 mL) and the rest of the
media
is replaced by sodium alginate containing ink. Furthermore, we have successfully proven that
Escherichia coli and Vibrio natriegens can survive inside the printed ink. Additionally we show
that
constructs in E. coli are indeed inducible inside our printed bio-ink when adding inducers later
to
a plate (see video below). All in our, our ink accommodates our engineered cells and is proven
to be
suitable for printing live bacteria.
3D Printing
We modified a commercial 3D printer and a laser CNC into a bioprinter [link to
hardware](Figure 4).
The laser CNC is difficult to use as a precision bioprinter since it uses voltage regulation
on a
brushless DC motor to control the flow rate. However, the 3D printer uses a stepper motor
(Nema 17)
which has a step of 1.8⁰. This means that we can achieve precise flow rates suitable for
printing
QR-codes with bio-ink.
Figure 4 - Our modified bioprinter.
Strain Design and Engineering
We have applied synthetic biology to achieve a secure QR code that would only appear
when the
correct inducers
are supplemented. We characterized and optimized the use of inducible promoters for V.
natriegens and E.
coli. Further, we investigated the use of different growing conditions in our model.
Promoters
pBAD in V. natriegens
We show that the commonly used part BBa_K808000 can be used for tight regulation in
V.
natriegens. Apart from low basal activity in the absence of inducer we
measured a 1127 time
increase upon induction with 0.5 (w/v) % arabinose. Consequently, pBAD works well
for the induced
regulation of gene expression in this organism. This on - off behaviour is ideal for
our application in
the biological QR-code in which we want the message only to appear in presence of
inducer.
Figure 5 – pBAD is a tightly regulated promoter that can be induced
by arabinose in V. natriegens. Fluorescence is normalized to OD600
and presented here on a logarithmic scale. The course of fluorescence is
presented for different inducer concentrations and at different time points.
Improved tetracycline inducible promoter using synthetic
promoter library (SPL)
Because of the outstanding importance of inducible phenotypes to our project we
optimized the tetracycline inducible promoter using a synthetic promoter library. We
have improved the part to have a high inducibility while maintaining a low basal
activity as well. Our improved version of this part exhibits a fold change of 34
upon induction.
Figure 6 – The basal level of reporter expression is comparable to
E. coli without the construct. Clone 6 from the synthetic promoter
library was induced with 250 ng/ml anhydrotetracycline. The used
fluorescence reporter is mCherry.
Growing Conditions and Viability
For our bio-ink it is important that the bacteria stay responsive. With our data and model we can show that LB supplemented with 20 g/l NaCl is the optimal choice as the media for growing V. natriegens. Check out our modelling page for more information!
Software
To show that our QR codes can be scanned, the following pictures show (on
the left) a picture of
plate in black and white taken with the biomolecular imager las-4000, then,
on the right, is the
(scannable) filtered image using our own software, which uses the shade of
the QR code to highlight
it and remove the rest of the background.
Figure 7 – edited image from Biological QR code using our software to make it scannable
The reason we used the las-4000 imager for this demonstration is due to the
reduction of glare and
other artifacts that would otherwise appear on the plate by using a phone in
normal light
conditions, due to the agar and the plate being glossy. Using a phone is still
possible, however,
the chances of detecting the QR code due to a lack of specialized software and
unfavorable light
sources makes are considerably reduces. For a more in depth description of the
software we developed
to improve readability of the QR codes please refer to the Software page under
awards.
