Our plasmid construct was assembled with a Golden Gate -inspired assembly method developed at our Department
of Life Technologies, Molecular Plant Biology unit. This assembly method was chosen because it has been
specifically designed for expressing proteins in Synechocystis sp PCC6803, and
allows us to make many variations of the same operon-based construct with relative ease. The system is now
under testing, and is derived from corresponding expression constructs previously assembled using BioBrick
–inspired subcloning (Thiel et al 2018; Nagy et al 2021). We did not have to order all of the parts for the
assembly ourselves but instead we were able to utilize our department's gene library, consisting of
characterized parts previously analyzed and validated in cyanobacteria. We only had to design and order our
merA and merB genes ourselves.
Our DNA elements were stored and amplified in commercial Escherichia coli plasmid
backbones (such as.) pEZclone-NRS-Amp-Blunt-HC plasmid [PK1] , and used for the Golden Gate assembly of a
cyanobacterial expression construct pDF. Our genes of interest ( merA Synechocystis
sp. PCC6803, merA Pseudomonas aeruginosa, merB Escherichia coli and merB Pseudomona aeruginosa) were deposited in the library in E.
coli plasmid backbones (Twist high copy AMP cloning vector with pMB1 origin of replicon).
The genetic elements used in the expression plasmid assembly, including the IPTG-inducible promoter PA1lacO-1
and the alternative RBS elements have been successfully used in cyanobacteria earlier (Thiel et al 2018; Thiel
et al 2019; Nagy et al 2021). Based on the previous information, these elements were expected to enable
high-level expression of the target proteins in Synechocystis.
In Silico assembly
We simulated the Golden Gate assembly with our parts in SnapGene (Figure 1) and made an example construct with merA Synechocystis (BBa_K4831000) and merB E.coli (BBa_K4831002) (Figure 2), to make sure that our designed sequences were correct for our golden gate assembly.
Harmonization
The target genes which were not native to Synechocystis sp. PCC6803 were codon-optimized to ensure proper translation in the host cell, as supported by literature (Rehbein et al. 2019). The optimization was done using CodonWizard (Rehbein et al. 2019) and was based on harmonizing the codon usage based on the relative presence in the genome. In the procedure, rare codons were not exclusively replaced by common ones to prevent changes in translation speed that could affect folding into the native active conformation. The harmonized codons in merA P. aeruginosa (Figure 3) merB E. coli (Figure 4) and merB. P. aeruginosa (Figure 5) , and] are shown in green, while the red and blue represent the codon usage in the donor organism, and in Synechocystis, respectively. The difference between the input and the output is not substantial in the codon usage between E. coli and Synechocystis (Figure 3), but is more pronounced in the case of merA (Figure 4) and merB (Figure 5) from Pseudomonas aeruginosa.
Table 1. A list of all Basic parts used in our project.
| Type | Part Number | Name | Description | Length |
|---|---|---|---|---|
| Basic | BBa_K4831007 | P_A1lacO-1-RiboJ | IPTG-inducible lac promoter variant containing a self-cleaving ribonuclease sequence for mRNA 5’UTR trimming. | 1782 bp |
| Basic | BBa_K4831004 | RBS_S3 | Translational control element RBS S3, containing the ribosome binding site and the start codon ATG. | 54 bp |
| Basic | BBa_K4831008 | RBS_S4 | Translational control element RBS S4, containing the ribosome binding site and the start codon ATG. | 54 bp |
| Basic | BBa_K4831005 | RBS_S5 | Translational control element RBS S5, containing the ribosome binding site and the start codon ATG. | 54 bp |
| Basic | BBa_K4831000 | merA_Synechocystis | Gene coding for MerA, which is a mercuric reductase. The sequence is from Synechocystis. | 1554 bp |
| Basic | BBa_K4831001 | merA_P. aeruginosa | Gene coding for MerA, which is a mercuric reductase. The sequence is from P. aeruginosa but codon optimized for Synechocystis | 1683 bp |
| Basic | BBa_K2308003 | sYFP2 | Gene coding for Yellow fluorescent protein, which can be utilized as a reporter gene. | 720 bp |
| Basic | BBa_K4831003 | merB_P. aeruginosa | Gene coding for MerB, which is alkylmercury lyase. The sequence is from P. aeruginosa but codon optimized for Synechocystis | 636 bp |
| Basic | BBa_K4831002 | merB_E. coli | Gene coding for MerB, which is alkylmercury lyase. The sequence is from E. coli but codon optimized for Synechocystis | 636 bp |
| Basic | BBa_K4831012 | EFE | Gene coding for Ethylene forming enzyme, which enables the conversion of intracellular 2-oxoglutarate into ethylene, and can thus be utilized as a reporter gene. | 1050 bp |
| Basic | BBa_K3257020 | Transcription Terminator | Sequence responsible for stopping the transcription of the gene. | 87 bp |
Table 2. A list of our designed Composite parts.
| Type | Part Number | Name | Basic parts in this composite part | Description | Length |
|---|---|---|---|---|---|
| Composite | BBa_K4831014 | IPTG inducible merA and merB (E. coli) expression device for Synechocystis sp. PCC6803 |
P1: BBa_K4831007
P2: BBa_K4831004 P3: BBa_K4831000 P4: BBa_K4831005 P5: BBa_K4831002 P6: BBa_K3257020 |
Composite for expressing MerA and MerB. The construct contains an IPTG induceable lac promoter variant, a RBS S3 followed by the merA (from Synechocystis), a RBS S5 followed by the merB (from E. coli) and a transcription terminator sequence. | 4187 bp |
| Composite | BBa_K4831013 | IPTG inducible merA and merB (P. aeruginosa) expression device for Synechocystis sp. PCC6803 |
P1: BBa_K4831007
P2: BBa_K4831004 P3: BBa_K4831001 P4: BBa_K4831005 P5: BBa_K4831003 P6: BBa_K3257020 |
Composite for expressing MerA and MerB. The construct contains an IPTG induceable lac promoter variant, a RBS S3 followed by the merA (from P. aeruginosa), a RBS S5 followed by the merB (from P. aeruginosa) and a transcription terminator sequence. | 4316 bp |
| Composite | BBa_K4831015 | IPTG indudable merA (Synechocystis) expression device with a EFE as a reporter gene |
P1: BBa_K4831007
P2: BBa_K4831004 P3: BBa_K4831000 P4: BBa_K4831005 P5: BBa_K4831012 P6: BBa_K3257020 |
Composite for expressing MerA and EFE. The construct contains an IPTG induceable lac promoter variant, a RBS S3 followed by the merA (from Synechocystis), a RBS S5 followed by the EFE and a transcription terminator sequence. | 4601 bp |
| Composite | BBa_K4831016 | IPTG inducible merB and YFP reporter device for Synechocystis sp. PCC6803 |
P1: BBa_K4831007
P2: BBa_K4831004 P3: BBa_K2308003 P4: BBa_K4831005 P5: BBa_K4831002 P6: BBa_K3257020 |
Composite for expressing MerB and sYFP2. The construct contains an IPTG induceable lac promoter variant, a RBS S3 followed by the sYFP2, a RBS S5 followed by the merB (from E. coli) and a transcription terminator sequence. | 3353 bp |
References
Literature references on the expression plasmid and genetic elements used for expressing MerA and MerB in Synechocystis sp. PCC6803 in the Aboa iGEM project 2023. Comments on why we used these references have been added.
Guerrero et al. 2012 is our original paper on the development of pDF and the comparison of various
promoters (including PA1lacO-1) in Synechocystis. This version of pDF has
been later used as a basis for developing pDF-CC used in the CyanoConstruct cloning system under
development. Most of our published studies base on variants of this plasmid, and the use of the
promoter PA1lacO-1.
Guerrero, F., Carbonell, V., Cossu, M., Correddu, D. and Jones. P. R. (2012) Ethylene synthesis and
regulated expression of recombinant protein in Synechocystis sp. PCC 6803. PLoS ONE
7(11):e50470.
http://dx.doi.org/10.1371/journal.pone.0050470
-----------------
Thiel et al. 2018 is our main reference to translational tuning; the generation and characterization
of the RBS library in Synechocystis; Rationalization for the need for
translational optimization; Uses pDF and PA1lacO-1, although the assembly system is based on a
BioBrick-inspired subcloning strategy that differs from the construct assembly Guerrero et al 2012, as
well as the Golden Gate cloning used in CyanoConstruct.
Thiel, K., Mulaku, E., Dandapani, H., Nagy, C., Aro, E-M., Kallio, P. (2018) Translation efficiency of
heterologous proteins is significantly affected by the genetic context of RBS sequences in engineered
cyanobacterium Synechocystis sp. PCC 6803. Microbial Cell Factories
17(1):34.
https://doi.org/10.1186/s12934-018-0882-2
-----------------
Thiel et al. 2019 is an example of generating operon-based multi-gene expression constructs in Synechocystis. Uses the same principle, pDF, PA1lacO-1 and selected RBSs (as
well as the assembly method) as described in Thiel et al 2018.
Thiel K, Patrikainen P, Nagy C, Fitzpatrick D, Pope N, Aro EM, Kallio P. (2019) Redirecting
photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC
6803 by the deletion of flavodiiron protein Flv3. Microbial Cell Factories, 18: 189.
https://doi.org/10.1186/s12934-019-1238-2
-----------------
Nagy et al. 2021 is a comparison of the replicative pDF to genome-integrated constructs in protein
expression in Synechocystis. Expands the discussion on the use and benefits
of the pDF plasmid.
Nagy, C., Thiel, K., Mulaku, E., Mustila, H., Tamagnini, P., Aro, E-M, Pacheco, C. C., Kallio, P.
(2021) Comparison of alternative integration sites in the chromosome and the native plasmids of the
cyanobacterium Synechocystis sp. PCC 6803 in respect to expression
efficiency and copy number. Microbial Cell Factories 20, 130.
https://doi.org/10.1186/s12934-021-01622-2
-----------------
Rehbein et al. 2019 is our main reference to codon harmonizing; how to change codons for efficient
translation without disturbing the kinetics of protein biosynthesis. Expands on our decision to
harmonize our genes from Pseudomonas aeruginosa and Escherichia coli.
Rehbein, P., Berz, J., Kreisel, P., & Schwalbe, H. (2019). "CodonWizard" - An intuitive software tool
with graphical user interface for customizable codon optimization in protein expression efforts.
Protein expression and purification, 160, 84–93.
https://doi.org/10.1016/j.pep.2019.03.018