Direct manipulation: engineering a cyanobacterium
The acclaimed exceptional growth rate of Synechococcus sp. elongatus UTEX 2973 was tested by the construction of a growth curve over a period of 17 days. The bacteria were cultured in a temperature room of 28°C, under atmospheric CO2 pressure and with a light intensity of 50 µmol/m².s, while being shaken at a constant rate of 200 rpm. It is important to note that these are not the optimal conditions mentioned by Yu et al. .
While it is clear that our growth curve is not as steep as the one obtained by Yu et al., this strain is proved to be remarkably easy to culture and exhibits rapid growth even under non-optimal conditions. It should be noted that it was not within the scope of the project to have these cultures growing as fast as possible but rather to manipulate the strain in a straight-forward manner.
As was mentioned in the project design, the S. elongatus UTEX 2973 strain caught our particular attention as a possible candidate to be used as chassis for sustainable biomanufacturing technologies. Working towards unlocking this potential, we engineered a new plasmid with our protein of interest, in this case, sfGFP, to be incorporated into the UTEX strain and serve as a reporter of the expression system.
Three signal peptides and the sfGFP insert were amplified by tail-PCR with overhangs compatible for Gibson assembly. Tail-PCR was verified using agarose gel electrophoresis. The resulting length of the signal peptides (SP) can be seen in Figure 2; those of modified GFP can be seen in Figure 3.
Following miniprep, the pAM2991 vector was digested by EcoRI, and subsequently by BamHI. Both digestions were verified by gel electrophoresis. The results are shown in Figures 4 and 5.
Gibson Assembly for the creation of the final construct, combining the pAM2991 plasmid, sfGFP, and signal peptides, was verified through PCR followed by gel electrophoresis. The results, showed the clear success of the assembly, which is shown in Figure 6.
Figure 6: Gel electrophoresis of insert validation performed using Colony PCR on colonies selected on streptomycin.
Unfortunately, we were not able to make the resulting pAM2991 vector integrate into the cyanobacterial genome. This conclusion was made after plating the cultures in both selective and non-selective media. While colonies naturally grew on a non-selective medium, those on a selective medium did not, suggesting a lack of antibiotic resistance and an absence of the plasmid. Consequently, two hypotheses can be made. A first possibility would be that the triparental conjugation lacked efficacy, meaning the plasmid might have never actually been in the cytoplasm of the cyanobacterium. Alternatively, it is possible that the plasmid did enter the cytoplasm, but that the mechanisms for genomic integration were absent.
Indirect manipulation: engineering cyanophage S-TIP37
Fragments were designed with overhangs compatible for integration into the S-TIP37 genome by Gibson Assembly. An enzymatic Cas9 reaction was used to cut the phage genomes in two parts, after which Gibson Assembly was performed.
Successful integration of the cassette into the S-TIP37 genome was verified with PCR followed by gel electrophoresis. Primers wrapping the insert locations were used for this (please see the Experiments section). The results of the gel electrophoresis are shown in Figure 7, from which it can be concluded that the engineered genomes were assembled as intended, implying the successful outcome of both the Cas9 digestion reaction and the Gibson Assembly. This is particularly the case for the three first locations, accounting for the substitution of the entire lysogeny cassette, the substitution of the integrase gene exclusively, and the insertion of cassette after the structural cassette. On the contrary, the fourth engineering strategy, insertion of the cassette after the last gene on the S-TIP37 chromosome, seemed to be less successful. A possible explanation might be that the last fragment of the S-TIP37 genome is relatively small compared to the other genomic pieces in the assembly, complicating the collisions of overhangs.
Due to malfunctioning growth of the cyanobacterial strain Synechococcus sp. WH8109, further experiments could not be performed. Hypothetically, the experiment would have continued as follows: the modified S-TIP37 genome would have been brought into the Synechococcus sp. WH8109 cytoplasm via electroporation. Once inside, the lytic cycle would have been rebooted, and new phage particles would be created, together with the production of our inserted fluorescent protein.
The fluorescent activity of the codon-optimized YFP was validated in E. coli, as is visible in Figure 8.
In order to verify if purified YFP would be comparable in fluorescence when expressed in a salty medium, an experiment measuring YFP fluorescence in varying salt conditions ranging from 0% NaCl to 10% NaCl over a period of three hours was performed. As shown in Figure 9, fluorescent activity did not seem to be affected by an increased salt concentration; subsequently, the salty medium or environment of the marine Synechococcus sp. WH8109 would not have been an obstacle in biomanufacturing of useful proteins.
Figure 9: Evolution of YFP fluorescence in different salt concentrations in time. Fluorescence was constant during this time period.
- J. Yu, M. Liberton, P. F.Cliften, R. D. Head, J. M. Jacobs, R. S. Smith, D. W. Koppenaal, J. J. Brand and H. B. Pakrasi, "Synechococcus elongatus UTEX 2973, a fast growing cyanobacterial chassis for biosynthesis using light and CO2," Scientific Reports, vol. 5, no. 1, p. 8132, July 2015.