Measurement
Most of the measurements performed in the results section were done by making creative use of
instruments and tools we had at hand (see Results). During our project we had to evaluate our
measuring procedures to see if they effectively represented reality. This was especially true because no
state-of-the-art procedures have been described when evaluating viability and inducibility inside gels.
Additionally, the execution of both the synthetic promoter library and CRISPR involved thorough
screening procedures. On this page we will show how some of our assays were developed and evaluated and
how these findings influenced our project.
Viability assay
A viability assay is generally used to determine the amount of viable cells inside a culture. For
our project it was important to determine the viability of Vibrio natriegens and Escherichia coli
inside our printed gels. The plate count method is the state-of-the-art procedure to perform a
viability assay. Bacteria, usually from a liquid culture, are serially diluted in milliQ, plated on
agar plates and visually counted after being grown overnight. To perform this assay, the bacteria
first have to be liberated from the hydrogel. A chelator such as ethylenediaminetetraacetic acid
(EDTA) or sodium citrate can be used to remove calcium ions from the hydrogel scaffold [1]. First,
the chelating effect of sodium citrate, a mild chelator, on the hydrogel and potential
negative effect on bacterial growth was evaluated. Solidified ink (1-2 % w/v sodium alginate) containing V. natriegens and E. coli was placed inside media supplemented with sodium citrate.
Growth and time taken for the gel to dissolve were evaluated and shown in table 1.
| Concentration citrate |
1.0 M |
0.5 M |
0.1 M |
| Time to dissolve of 2% alginate gel |
3 min |
4 min |
12 min |
| Time to dissolve of 1% alginate gel |
5 min |
5 min |
7 min |
| Growth at 2% alginate gel |
No |
Yes |
Yes |
| Growth at 1% alginate gel |
No |
No |
Yes |
Table 1 – impact of sodium alginate on growth of E. coli and V.
natriegens.
What can be seen is that at high concentration of sodium citrate, no growth can be observed.
This hints on a negative effect of sodium citrate on growth. Furthermore, complete chelation
of the hydrogel scaffold still occurs in about 12 minutes when using 0.1 M. This result prompted us to
dissolve alginate hydrogels in 0.1 M sodium citrate to minimize the negative effect of
sodium citrate on the outcome of the viability assay.
Next, a trial was performed to evaluate the viability of both organisms in the printed
hydrogels. However, when plates were counted, no colonies were observed on plates beyond
the 10 times dilution for V natriegens. Since V. natriegens is a halophile and generally
grows in high NaCl containing environments, we hypothesized that by adding the bacterium
in an environment without salt, the sudden and drastic change in osmotic pressure
critically influences the bacterium. Indeed, when Vibrio was serially diluted in
duplicate in sterilized 30 g/L NaCl solution, visible colonies could be counted and
viability was assessed in multiple conditions (Figure 1).
Figure 1 – A, Viability assay on a print plate with nutrients at room temperature (RT), B, Viability assay on plate without additional nutrients, stored in the fridge, C, Viability assay on plate without extra nutrients, stored at room temperature
Plates that had more than 400 colonies were not counted because of the sheer
amount off cells. Plates with less than 25 colonies were also discarded because
the amount of bacteria on these plates is too few to rely on. By taking all
these precautions, we believe to have developed an accurate and efficient
protocol for determining bacterial viability inside printed calcium alginate
hydrogels.
Interestingly, this outcome changed how we would handle V. natriegens throughout the whole project. Every time we diluted or re-suspended V. natriegens, the salt
concentration was considered.
Determining inducibility
Again, no standard procedure exists for determining fluorescence in printed
ink on plates. Fluorescence is commonly used to quantify responses of for
example promoters and more complicated circuits. For this application
fluorescence is usually measured in a plate reader. For our project, we had
to determine inducibility inside printed ink. Plate readers are not set up
to perform fluorescence measurements in solid media. Therefore, the Typhoon
FLA 9500 biomolecular imager was used to image the plates with the ink. This
machine can use a set of lasers and filters to image gels and western blots.
Due to its high sample throughput we were able to simultaneously image all
of our plates in one experiment, allowing for a comparative quantitative
analysis. A shape was drawn to select the region of interest three times to
increase the accuracy of the protocol and reduce observational bias (Figure
2). Fluorescence was quantified by selecting the region of interest in
triplicate and using the measurement command for average intensity in imageJ. We
believe that by imaging and evaluating the plates in this way, an accurate
quantitative assessment can be made (figure 3A & 3B).
Figure 2 – Area selection for average intensity calculation in imageJ
Figure 3 - Quantified analysis of fluorescence measurement for both inducibility experiments (link to results). A, inducible GFP experiment. B, inducible RFP experiment.
Screening for synthetic promoter library and CRISPR
Our designs of the synthetic promoter library (SPL) and CRISPR included
a thorough and multiple step screening process. For the
SPL the
induction of fluorescence was measured in multiple microtiter plate
reader experiments. After transformation with the plasmids containing
the randomized promoter grown clones were picked and cultured in liquid
medium. The grown cultures were used to start new culture with and
without the inducer. All clones were tested for mCherry expression after
nine hours of incubation. The exact numbers of clones handled are
featured in the following table:
|
pTET |
pBAD |
| E. Coli |
161 |
84 |
| Total |
245 |
| V. natriegens |
21 |
5 |
| Total |
26 |
| TOTAL |
271 |
Table 1 – impact of sodium alginate on growth of E. coli and V.
natriegens.
This initial screen gave a first impression of whether the created
construct was inducible or not. Based on this measurement 10 clones
from E. coli experiments were included in a second screening
process and
where analyzed in a similar manner. This step revealed our optimized
part BBa_K3171173 which showed a 34 fold increase in
fluorescence upon induction.
In order to execute the CRISPR mediated knock out 3 gRNA
target sites and 3 homologous parts were designed resulting in 9
different reaction mixes. After transformation of the needed
machinery into E. coli and V. natriegens, they were
plated on plates
containing the selection marker kanamycin and the Cas9 inducer
mannose amounting to a total of 18 plates. From each plate that showed
growth within 48 h clones were picked and checked for histidine
auxotrophy. In total 80 were plated on minimal media with and
without histidine supplementation, 40 per organism. Clones
growing only in presence of histidine are auxotrophic. However,
this screening revealed that no auxotrophy was achieved so the
protocol should be revised and optimized in the future.
