An important principle in synthetic biology is the engineering cycle, also known as the Design-Build-Test-Learn (DBTL) cycle. In DBTL cycles, engineering principles are used to design, build and test biological systems, and to learn from these results to improve the initial design. DBTL cycles are repeated until a built biological system fulfills the intended functionalities. Instead of a succession of cycles, we rather see this process as a spiral where each additional DBTL cycle gets smaller and smaller and progressively converges to the target system. We therefore introduce you to the Spiral of Engineering Success!
Click on the spiral to take you to the respective cycle!
In the course of our iGEM project, we went through many engineering cycles, be that in the aerogel production, in the recombinant proteins production or in the coating of the aerogel with the proteins. We decided to dedicate this Engineering Success page to the proteins expression and purification, which has been an extensive process not as straightforward as imagined. Eventually, we were able to successfully build and test one new basic part and two new composite parts that we added to the iGEM registry: mSA-N[AS]4C-CBD-10xHis (BBa_K4439007), SR-Avitag-10xHis (BBa_K4439015) and mSA-GFP-CBD-10xHis (BBa_K4439013).
Engineering Cycle 1
This DBTL cycle is about our initial plasmid design and the first building and testing of the system we aimed to create. During those first tests, we encountered unexpected issues that we troubleshooted in the next DBTL cycles.
When designing the plasmids encoding the fusion proteins we wanted to coat on the cellulose aerogel, we used engineering and synbio principles such as modularity, the use of previous iGEM parts and standard expression system, as well as the insertion of restriction sites between each gene sequence to enable flexibility in the design even after building the DNA parts. If you want to read more about the design process and the engineering considerations we had, or look at our designed plasmid maps, check out the Engineering choices in our Design page. We designed three parts that were inserted in a pET28a vector and tested: mSA-silk-CBD-10xHis (01a), SR-Avitag (01b) and mSA-GFP-CBD-10xHis (03a). The proteins corresponding to these parts are shown below.
For the building of the biological system, a DNA synthesis supplier produced our designed plasmids 01a, 01b and 03a. After receiving the ordered plasmids, we transformed BL21(DE3)pLysS E. coli competent cells with these plasmids. After having sequenced the amplified plasmids for quality check, we induced protein expression in the same E. coli strain by adding IPTG.
We sequenced the three amplified plasmids and discovered on each plasmid added sites on the N-terminus that were in frame with our fusion proteins, in particular a 6xHis-tag. There were in total 100 bp that we did not expect to have within the plasmids sequence. We also sequenced the original plasmids provided by the company that had not been amplified in E. coli, and we obtained the same results.
We wondered if the additional 6xHis-tag on the N-terminus would interfere with the His-tag purification since we already added a 10xHis-tag on the C-terminus of the fusion proteins. We decided to still continue with these plasmids to test if the purification of such proteins was still possible and if the function of the proteins was not impared by these sites. Protein expression in BL21(DE3)pLysS and His-tag purification with the MagneHis purification kit resulted in no purified protein, except for 03a in little amounts, and a contaminant protein was spotted in the elution. We tested the expression and purification with a SDS-PAGE and a Western blot.
From our sequencing analysis, we learned that those unexpected sites in the N-terminus of the proteins were from the pET28a backbone used by the DNA synthesis company, the pET28a backbone from AddGene used for the plasmids design did not contain those sites.
Regarding the purification, troubleshooting led us to the conclusion that the contaminant protein was coming from the strain BL21(DE3)pLysS we were using since this protein also appeared in the elution of untransformed bacteria and non induced bacteria. Contaminant proteins in the elution usually occur when the protein of interest is expressed in very low amounts.
BL21(DE3)pLysS expresses the T7 lysozyme which reduces basal expression of the T7 RNA polymerase, avoiding leaking expression of the gene of interest in the absence of IPTG, but a side effect is that it can also reduce the expression after induction, resulting in lower protein yields1. This might explain why we had a low expression of recombinant proteins.
Recap of Cycle 1
In this first engineering cycle, we realized that the built plasmids did not match the designed plasmids and that proteins were expressed in too low amounts. We troubleshooted the low expression issue in Engineering Cycle 2 and we cloned the plasmids to remove the unexpected sites in Engineering Cycle 3.
Engineering Cycle 2
To troubleshoot the low recombinant protein expression, we switched the bacterial strain and followed a different His-tag purification protocol.
Since the recombinant proteins were not sufficiently expressed in the strain BL21(DE3)pLysS and based on literature research, we decided to change the bacterial strain used and tested two others: BL21(DE3) and Rosetta E. coli strains. Regarding the problem of the contaminant protein, we opted for a different His-tag purification protocol that we had already successfully done with another protein as a training at the Protein Production and Structure Core Facility (PTPSP).
We transformed BL21(DE3) and Rosetta E. coli strains with the plasmids 01a (silk fusion protein), 01b (SR fusion protein) and 03a (GFP fusion protein) synthesized by the DNA supplier. The transformation was successful in BL21(DE3) for the three constructs but in Rosetta it only worked for 01a. We induced expression of the recombinant proteins with IPTG.
This time, we were able to purify the three fusion proteins, however the purification did not go smoothly because most of the His-tagged proteins were stuck on the Ni-NTA beads even with high amounts of imidazole in the elution buffers. We had to go up to 5M of imidazole to remove almost all the proteins from the beads, whereas the usual elution buffer contains 300 mM imidazole). We tested the expression and purification with SDS-PAGE gels and Western blots.
Most likely, the His-tags on both sides of the fusion proteins (6xHis-tag on N-terminus and 10xHis-tag on C-terminus) were disturbing the His-tag purification, requiring a larger amount of competitor (imidazole) to elute them from the purification column. It might be that both His-tags were binding the column, meaning that to elute the fusion proteins, two interactions had to be broken instead of one. Since high amounts of imidazole could be too harsh conditions for the proteins, even if temporary, we had to find a way to reduce the imidazole concentration needed for His-tagged proteins elution.
Recap of Cycle 2
In this second engineering cycle, we successfully expressed and purified the three recombinant proteins 01a (silk fusion protein), 01b (SR fusion protein) and 03a (GFP fusion protein), however the purification process had to be improved because the amount of imidazole used for the elution was abnormally high.
Engineering cycle 3
Given the complications encountered during the His-tag purification of the plasmids with the additional sites, we decided to clone these plasmids to remove the 100 bp added to the constructs.
In order to obtain a smoother purification protocol requiring reasonable amounts of imidazole for the protein elution, we designed a cloning strategy to remove the unwanted sites from the three plasmids received by the DNA synthesis company, in order to recover the plasmids we originally designed. We used the PCR-KLD cloning method: we amplified by PCR the part of the plasmids we wanted to keep (everything except the additional 100 bp), and after the PCR we used a KLD enzyme mix allowing the fast and efficient phosphorylation and circularization of the PCR fragments without the need of using restriction enzyme digestion. We used the NEBaseChanger cloning website to design the KLD primers. We did a in silico cloning by simulating the different cloning steps with SnapGene, and the resulting plasmids were exactly matching with the originally designed plasmids, with the addition of a PmeI restriction site within the NcoI restriction site.
We performed the PCR-KLD cloning and the transformation of NEB 5-alpha E. coli cells with cloned plasmids was successful. During this build stage, we went through a small DBTL cycle within it because we had to troubleshoot the PCR step that was not working.
We sequenced the plasmids cloned by PCR-KLD, after amplifying them in NEB 5-alpha E. coli cells. We also performed a restriction analysis of the KLD plasmids on an agarose gel to confirm the sequencing results. Plasmids 01b (SR fusion protein) and 03a (GFP fusion protein) had successfully been cloned - removal of the unwanted sites and addition of the PmeI restriction site were confirmed - but the 01a (silk fusion protein) PCR-KLD cloning did not work: a part of the silk sequence had disappeared (~800 bp), leading to a shortened gene insert.
Engineering cycle 3’
Since the PCR-KLD cloning did not work for the silk plasmid 01a, we troubleshooted the cloning of this plasmid by designing another cloning strategy.
Troubleshooting the cloning of the silk plasmid
The PCR-KLD cloning did not work for the silk fusion protein gene (01a) because of the repetitive modules present in the silk sequence that most likely formed a secondary DNA structure interfering with the PCR by hindering some DNA sequences that did not get replicated by the DNA polymerase.
We therefore designed a new cloning strategy that did not require a PCR step. Luckily, in the 01a plasmid sent by the DNA synthesis supplier, two NcoI restriction sites were flanking the 100 bp added sites. We thus thought about performing a simple NcoI digestion of the 01a plasmid followed by ligation, without having to on one hand digest a plasmid backbone and on the other hand digest the silk fusion protein insert. We did a in silico cloning by simulating a restriction fragment deletion with SnapGene and aligned the expected result with the originally designed plasmid and the one received by the DNA synthesis company: this cloning strategy would enable to remove the unwanted sites but not to insert a PmeI restriction site as originally desired, a NcoI scar would remain instead.
We performed the cloning we had designed: a NcoI double restriction digestion of the 01a plasmid, followed by a plasmid purification from agarose gel and ligation. We then successfully transformed BL21(DE3) with the ligation mixture.
Sequencing results revealed that we successfully cloned the 01a silk plasmid: the unwanted sites were gone but PmeI had not been inserted, as expected with this cloning method.
At the end of this troubleshooting cycle 3’, we successfully cloned the three plasmids and got rid of the additional sites on the N-terminus of the fusion proteins. 01b (SR-Avitag) and 03a (mSA-GFP-CBD) were obtained with the PCR-KLD cloning strategy, whereas 01a (mSA-silk-CBD) was obtained with the NcoI cloning strategy.
When testing these new constructs in the usual strain BL21(DE3) for protein expression, we realized that the GFP (03a) and SR (01b) fusion proteins were not expressed anymore upon IPTG induction, while the silk (01a) fusion protein was expressed. We were able to purify 01a protein in high amounts with 250 mM imidazole in the elution buffer, a smaller and more normal concentration as previously.
From these results, we concluded that the NcoI cloning strategy for the silk plasmid 01a led to a functional biological system enabling the production of the mSA-silk-CBD protein needed to form a waterproof biofilm to coat on the cellulose aerogel. The removal of the 6xHis-tag and other tags on the N-terminus of the silk fusion protein indeed resolved the issue of high imidazole concentrations required for the elution, as hypothesized. However, the PCR-KLD cloning strategy did not enable the successful expression of the SR-Avitag and mSA-GFP-CBD proteins although the plasmids were validated by sequencing. The cause of this failure was quite obscure, even the experts from the PTPSP were surprised about it. The most probable reason for the absence of protein expression was the number of bp between the RBS and the first ATG start codon (Methionine), since the newly cloned 01a plasmid kept the same number of 6 bp because we could not add the PmeI site, whereas the 01b and 03a plasmids cloned with PCR-KLD got 8 additional bp (14 bp in total) between the RBS and the first start codon due to the PmeI site addition.
Recap of Cycle 3
In this last full engineering cycle, successful cloning of the plasmids 01a (mSA-silk-CBD), 01b (SR-Avitag) and 03a (mSA-GFP-CBD) was achieved: the 100 additional bp that impeded the His-purification were removed from the plasmids. However, only the 01a newly cloned plasmid led to a successful protein expression and smooth purification. The new SR and GFP fusion proteins could not be expressed in BL21(DE3).
We unfortunately did not have time to troubleshoot this expression issue for the SR and GFP fusion proteins, but if we have had more time we would have played with the number of bp between the RBS and the first START codon, to be the same as the new 01a plasmid and the other plasmids provided by the DNA synthesis supplier. We could also try different IPTG induction levels to find optimal protein expression.
Overall, despite all the challenges we faced during our iGEM project, we were still able to purify the fusion proteins we wanted for the aerogel coating after two iterations of the engineering cycle. The additional third cycle was for optimization of the system, which eventually was a success for one of the three constructs, the silk fusion protein. The successful optimization of the SR and GFP fusion proteins should require one additional engineering cycle for which we gave improvement suggestions.
In conclusion, we were able to successfully build and test one new basic part and two new composite parts that we added to the iGEM registry.
Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systemsApplied Microbiology and Biotechnology, vol. 72, no. 2, pp. 211-222