Engineering success
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To create the final plasmid for our recombination system Lambda RED, we had to overcome several obstacles before we obtained the desired result. Read below all about our struggle, our quality controls, our (partly self-inflicted) stumbling blocks and how we finally succeeded!
A great innovation or a success in any field is usually no coincidence but the result of an open mind, critical thinking and the will to start over if necessary. Often, the innovator has to go through parts or even the whole design cycle repeatedly, he/she has to Research → Imagine → Design → Build → Test → Learn → Improve → Research... multiple times before he can claim the desired outcome. Synthetic biology is no exception in this, and so we also faced some situations in our iGEM project where we had to try more than one approach until we succeeded. Our most important example of this cycle is probably the cloning of our Lambda RED recombination plasmid:
Research(1)
The main goal of our iGEM project is to create our genetically engineered T7 phage PHANGEL through genomic recombination. To achieve this, we decided to use the Lambda RED Recombination System. We did extensive research on the mechanism. More details to the system can be found in our project description. In a nutshell, it consists of three proteins called Exo, Beta and Gam each having a different function in the recombination process. To have them all in one plasmid in our production cell line seems the right idea to ensure that the recombination goes as smoothly as possible in the bacterial cells. As phages are a kind of virus, they can only replicate within their respective host organism and often even harness the replication machinery of the host to achieve this. Hence, we need a bacterial host strain, called the production cell line, in which the homologous recombination can take place.
Due to toxicity of the expressed proteins, the addition of lac repressor is necessary in the recombination plasmid. We, therefore, decided against the normal T7 promoter to avoid leaky expression of the genes and used a T7 promoter combined with a Lac operon as a Lac repressor is constitutively active in our production cell line E.coli BL21 (DE3). Expression of the recombination proteins would now only happen in the presence of IPTG.
Imagine (1)
Since our PIs have a lot of experience with Golden Gate Cloning, we chose this as our method to assemble the recombination plasmid with the genes for all three proteins. Even though the team members had not so much experience with it yet, this posed the advantage to ask for help and materials on short notice. This allowed us, e.g. to save resources on our IDT sponsorship as the required plasmid backbones were already available for us.
Design (1)
Once we decided on a method, we planned the cloning of our plasmids. We attached the necessary fusion sites for Golden Gate Cloning in silico to the gene sequences of the recombination proteins and ordered them from IDT. We needed three separate cloning steps to obtain the finished plasmid as we needed to attach the promoter and a terminator sequence to obtain the three open reading frames. Furthermore, we wanted antibiotic resistance as a selection marker for the successfully transformed cells after cloning. To achieve this, we chose a backbone containing a kanamycin resistance gene.
Build (1)
We started with the first cloning step of the three separate genes into three separate Golden Gate compatible backbones. These first backbones also contained a kanamycin resistance cassette as a selection marker. Beside this selection marker, we wanted to make sure that our genes were inserted flawlessly.
Test (1)
To control if our genes were inserted into the first backbone, we performed a MiniPrep to isolate the plasmid DNA, restriction digested it with Bbs1 and performed a gel electrophoresis. This first electrophoresis showed great results, however, we also wanted to check if our inserts were free of point mutations so our proteins would be expressed correctly. Hence, we sent the plasmid DNA to be sequenced. The results were quite mixed. Exo and gam were flawless and we could move on with them to clone them into the second plasmid backbone where they would be combined with their promoters and terminators. The result for beta, on the other hand, was not as expected. The sequencing result did not align at all with the original sequence in our computers.
Learn (1)
After a few experiments in SnapGene to find the error, we realised that the plasmid insert we thought was beta, aligned perfectly with the sequence for exo.
Improve (1)
We thought we made a pipetting error, using the gene for exo twice in our cloning experiment and now cloned the “right” beta into the first backbone again.
Test (1.2)
Again, we wanted to check the sequence for beta for point mutations and sent it to be sequenced. The results this time were disappointing: the sequence did once again not align with our in silico sequence for beta but with exo! A quick look into the order from IDT proved that we had, in fact, made an error not while pipetting but while ordering and had ordered the sequence for exo twice instead of exo and beta.
Learn (1.2)
We learned the hard way that Copy-Pasting sequences is not free of errors and to double check if the sequences are correct before ordering next time.
Improve (1.2)
We now needed to make sure to receive the “real beta” quickly to not lose any more time. In this improvement session we also considered ordering the finished plasmid to spare us the cloning work.
Research (2)
Ordering the whole plasmid, however, turned out to be impossible in the remaining time of our project: most sellers had a delivery time of four to six weeks. By then our lab time would have already been over so this was not an option.
Imagine (2)
We now needed to find a way to complete our GoldenGate Assembly into the third backbone without losing time having to start all over again.
Design (2)
We came up with the idea to create a version of the beta sequence that already included the promoter and terminator sequences and had the fusion sides of the second backbone. With this design, beta would be completely ready to be combined with exo and gam in the third backbone. While we waited for this new beta construct to arrive, we had enough time to clone exo and gam together with the promoters and terminators into their second backbone.
Build (2):
When beta arrived, we were finally able to combine all three gene constructs in their final backbone.
Test (2):
A gel electrophoresis proved that all the inserts were there and had the right size. However, since we used Golden Gate Cloning, our parts were not compatible with the registry yet.
Learn (2):
We got to know the systematics of the iGEM BioBricks in more detail and grew familiar with new cloning systems.
Improve (2)
We adapted the parts to make them compatible with the iGEM registry format and uploaded them so other people will be able to access them for future projects. You can check them out here:
BBa_K3514001
BBa_K3514002
BBa_K3514003
BBa_K3514004
Next steps:
Even though we were not able to produce our recombinant bacteriophage in time, we still planned the next steps in our experiments with the phage. Read below what we had planned and partly already prepared as further experiments for our project:
We planned the transformation of our bacterial producer cell line E.coli BL21 (DE3) with a double antibiotic resistance (ampicillin on the capsid backbone (see safety mechanism ) and kanamycin on the recombination plasmid backbone) to be able to select the cells carrying both plasmids for our recombination experiments. We tested two different methods for the transformation of our linear genes (phage genome and LPS binding proteins) into the producer cell line: heat shock transformation and electroporation. We decided on electroporation as this is also recommended in the literature and, based on this decision, we tested different phage DNA concentration for this transformation method as we read a too high DNA concentration is as harmful as a too low concentration. We also tested if the addition of IPTG after the transformation would make a difference in the expression of viable phages from the transformed DNA as the T7 phage in E.coli BL21 (DE3) is controlled by a Lac operon. All these tests were evaluated through phage plaque assays. The results, however, were inconclusive and if we had had more time, we would have conducted these experiments again.
We also calculated in a simple model at what time after inoculation phages needed to be added to a bacterial culture to produce the highest number of phages in the shortest time. These efficiently grown phages were meant to be lysed to use their isolated DNA for recombination experiments later.
We designed our gene sequences for the protein and peptides we wanted the recombineered phage to produce. Their sequences needed special recombination sites to be inserted into the correct place in the genome and their own promoters and terminators. Furthermore, we added His-Tags to each protein or peptide to be able to isolate it after translation. After their design in silico, we ordered them and amplified them via PCR. We purified them using the NEB Monarch PCR & DNA Cleanup Kit (5 μg) to get rid of any remaining PCR components (salts, enzyme etc.) that could impair the transformation.
We researched and prepared protocols for the recombination experiments with the phages.
Additional to the design of the protein and peptides, we designed special primer pairs for each gene sequence: one primer pair flanked the whole inserted gene, the other one had one primer flanking and the other primer inside the inserted sequence. We wanted to use them to be able to screen for correctly recombineered phages later. Furthermore, we prepared the protocols for the His-Tag purification and already considered conduction of Western Blots and ELISAs to prove our concept was working.
If all of this were successful, we learned in one of our expert interviews that we could test the ability of our protein and peptides to neutralise LPS in an immunoassay with macrophages and/or neutrophils.
Conclusion:
As you can see, we had a lot planned for this summer. Unfortunately, our lab time was very restricted due to Covid-19 safety measures and we spent a good portion of the time left trying to clone our recombination plasmid correctly. We were wise to include all these testing and quality control steps described above as we could have ended up with a seemingly flawless but unfunctional recombination plasmid that included exo twice instead of exo and beta. Even though we did not get as far as we had imagined with our project in the scope of the iGEM competition, we are certain we learned a lot by following the engineering design cycle and are proud of our accomplishments. Maybe our project idea can form a base for further research on the topic of engineered phages.
