Introduction
During our project, we faced numerous challenges in the engineering part. These obstacles sometimes proved to be hard ones to overcome, but we can say it honestly that we did not step back. In this short review, we present our troubleshooting cycles through which we have learned: biology is challenging, and often the simplest designs can fail. With creativity and rational design, we managed to overcome several barriers standing between us and success.
BLADE
1. Cycle: False positive diagnostic digestion → Improved plasmid isolation
Research
In our project, the cytotoxic protein needed to be produced only in response to an external stimulus, in our case, blue light. The system is based on a promoter that can be regulated by a protein that dimerizes in response to blue light, called BLADE and described by Romano et al. [1]
We planned to test this design in the BL21 strain and characterize it by illuminating the bacteria with blue light and examining protein production.
To facilitate this, we designed not only the CylA protein of our choice but also a model construct using a reporter protein, fluorescent mCherry, behind the promoter. This, we could check the promoter's function by measuring the fluorescence.
Imagine
This construct was designed to be cloned into a pEV vector using conventional cloning techniques. For this purpose, a bacterial codon-optimized sequence was ordered from IDT using XbaI and XhoI enzyme cleavage sites interspersed.
Design
We decided to commission the synthesis of the BLADE to construct the downstream protein coding region from IDT in one step due to the short time available and to minimize the upstream risks. Thus, with a simple single-incision ligation, our plasmid can be created. In addition, several members of the team had extensive experience in this cloning technique.
Build
For cloning, we digested the vector and the ordered inserts with the appropriate restriction enzymes and performed the ligation with T4 ligase.
Test
Plasmids were isolated and digested with XbaI and XhoI enzymes, and gel electrophoresis was performed on the samples. Although faintly visible on the gel (because we did not digest enough DNA), the insert was visible in some clones. So we selected these and started protein production studies with them. However, no results were obtained. So as a control, we re-isolated the plasmid and digested it, and this showed that the insert was not present in the plasmid, so it was clear why we did not get any results from the protein production assay.
Learn
We have learnt that an accurate gel image is essential for a proper conclusion because if we go ahead with an uncertain result, it means a lot of extra work and time for us.
Improve
By isolating the plasmid, more attention was paid to obtaining a sample of sufficiently high purity and higher concentration. This was significant because the low plasmid concentration in the first attempt meant that we did not digest enough to see the results properly on the gel.
Test
We did not get colonies on the plate.
2. Cycle: No colonies after transformation → New vector
Research
Behind the lack of colonies, we first suspected that either the inserts or the vector were not digested properly. Digestion of the inserts is difficult to check, as only a few bases with a few overhanging ends are cleaved. In the case of the plasmid, this is easier to check, and we could test the ability of the enzymes used to work.
Imagine
In the pEV vector, the XhoI and XbaI cleavage sites are very close, so it is not so clear in the gel image whether it has been digested properly. So we looked for a vector where if we digested with the two enzymes, a clearly visible fragment would fall out.
Design
We chose the pETARA vector and planned to digest it for cloning, as a fragment of around 1000 bp is lost from this plasmid during XbaI and XhoI digestion.
Build
We have digested the pETARA vector. The gel clearly showed the fragments that had fallen out, so we could be sure of proper digestion. The previously digested inserts were ligated into the vector.
Test
We did not get colonies on the plate.
Learn
We saw that the vector was digested properly, so the problem is not with the enzymes and not with the vector digestion.
3. Cycle: No colonies after transformation → Change of used cloning bacterial strain
Research
Since we again did not receive any colonies, we took a look at what the problems might be. It was suggested that there might be a problem with the competence of the competent cells, so we tried to eliminate this.
Imagine
Since we found that the DH5α cells were not as competent as Xl1Blue, we switched to this one.
Build
We performed the cloning according to the previous protocol with a freshly digested vector, but in the end, the transformation was performed in Xl1Blue cells.
Test
We did not get colonies on the plate.
Learn
From this, we learned that the problem may not be with the competence of the competent cells. (Also, competent cells from the same stock have been used by others and worked well for them.)
4. Cycle: No colonies after transformation → Introducing extra nucleotides to the close of the DNA fragments
Research
After several failed transformations, we checked the sequences again and asked our mentors what the problem might be. They pointed out that we hadn't left enough nucleotides at the beginning of the cleavage sites, so it was probably due to the low efficiency of digestion of the insert.
Imagine
To solve this problem, we had to introduce extra nucleotides in front of the cleavage sites in the sequences.
Build
We designed primers that contained extra nucleotides beyond the cleavage sites, allowing us to use PCR to extend the sequence of the inserts and thus increase their digestion efficiency.
You can find these primers in the Registry as Forward primer for BLADE (BBa_K4375024), Reverse primer for J23101-BLADE-ClyA (BBa_K4375025), and Reverse primer for J23101-BLADE-mCherry (BBa_K4375026).
Test
After the PCR reaction, a product of the correct size was obtained, purified from gel digest, and ligated into a freshly digested pETARA vector. However, by the next day, we still had no colonies on the plate. We set the ligation reaction aside and ran it to see if we could see any occluded vectors. This gel pattern was as expected.
Learn
At this point, we had tested everything we thought might be causing the problem (enzymes, inadequate digestion of vector or inoculum, competent cells not functioning properly, antibiotic degradation). So, in consultation with our advisor, we took it to another lab to see if we could get a hit using the tools they had.
5. Cycle: No colonies after transformation → New lab
Build
We took all the samples to the Vascular Research Group's lab and went through the entire process again, from PCR amplification of the inserts to starting the work.
Test
Here we obtained colonies, and after plasmid isolation, we performed diagnostic digestion. The gel electrophoresis confirmed the presence of the correct insert in the plasmid, and the presence of the fluorescent protein was detected.
So after a long time, we finally achieved success.
Test+1
As a good researcher, he did not leave us alone as the reason for our failure. It was suspected that perhaps one of the batches of LB agar was not good. To check this, we spotted competent cells transformed with a plasmid containing the appropriate resistance on one of the plates. Since no colonies were found here, we concluded that the failed agar might indeed be the cause of our ordeal.
Hardware
1. Cycle: How to test the bacterial construction as early as possible → Modular illumination device consisting of home lighting elements
Research
The BLADE expression system is blue light-inducible (~450 nm) therefore an appropriate irradiation setup is needed for testing our constructs.
Imagine
We have listed our requirements for the irradiation setup: (1) the LED must emit light at the optimal wavelength, (2) it can be cooled, (3) it can be put into an incubator, (4) it needs to have a dimmer function, and (5) it must be safe. To test our bacterial construction as soon as possible, we first thought about a device that would be quick to assemble and easy to use.
Build
We have built our first irradiation setup with components that can be found in a household and easily obtainable in a shop. The unique illuminating area is a plastic cylinder, the blue LED strip is attached to the wall which is cooled by a PC fan.
Test
The illumination device was successfully installed and shaken in the incubator. During the experiment, the samples did not overheat, so air cooling was suitable. The first test reactions showed that the wavelength was appropriate, and the experiment was successful.
Learn
This device was adequate to draw significant conclusions about how our system works. We understood that it was not suitable for more complex experiments or testing its interaction with mammalian cells.
2. Cycle: How to carry out mammalian cell illumination experiments → Creating RaccOpto
Research
From the drawbacks of the previous hardware, we knew that our illumination device had to be programmable. Furthermore, we needed multiple channels if we wanted to illuminate a 96-well plate so that we could test it with different illumination programs per row or column.
Imagine
Since the literature and commercially available devices did not meet all our requirements, we wanted to assemble our lighting device from modular parts that could be bought in stores. We aimed to make installation and repair as easy as possible.
Build
As we found suitable modular units for each of the hardware tasks, our main task during the construction was to create a unique lighting area. To dissipate the heat, we placed the blue LEDs in 96 holes drilled in a 6 mm thick aluminum sheet, cooled by a PC fan.
Test
Fearing hardware incompatibility of some parts, after assembly, we were pleased to find that our system worked correctly and programming was successful. Unfortunately, we found some columns did not have all LEDs lighted up.
Learn
Finding the fault, we discovered that some LEDs have an incorrect plastic coating due to a factory defect and become short-circuiting when in contact with the aluminum plate. Paying close attention to this, we corrected the illumination area and created a per-pole controllable device in addition to a per-row controllable one.
[1] Romano, E.; Baumschlager, A.; Akmeriç, E. B.; Palanisamy, N.; Houmani, M.; Schmidt, G.; Öztürk, M. A.; Ernst, L.; Khammash, M.; Ventura, B. D. Engineering AraC to Make It Responsive to Light Instead of Arabinose. Nat. Chem. Biol. 2021, 17, 817–827. https://doi.org/10.1038/s41589-021-00787-6.