Summary
Before assembling our methanol condensation genes in one genetic construct, we wanted to obtain the required genes separately in a pSB1C3 standard BioBrick vector.
| BioBrick |
enzyme |
gene |
sequence length [bp] |
source organism
|
| BBa_K1585210] |
methanoldehydrogenase 2 |
mdh |
1219 |
Bacillus methanolicus MGA3
|
| BBa_K1585211 |
3-hexulose-6-phosphate |
hps |
697 |
Bacillus methanolicus MGA3
|
| BBa_K1585212 |
6-phospho-3-hexuloisomerase |
phi |
616 |
Bacillus methanolicus MGA3
|
| BBa_K1585213 |
phosphoketolase |
xpk |
2539 |
Bifidobacterium adolescentis
|
As the coding sequences that are required for methanol condensation come from different organisms, they first had to be codon optimized. As the next step, extensions at both ends were designed to make the genes consistent with our cloning strategy. After amplification, the BioBrick vectors were assembled via restriction and ligation as well as CPEC, respectively.
in silico work
Bacillus methanolicus is an organism that can naturally metabolize methanol and carries the coding sequences of mdh, hps and phi in its genome [1]. We decided to take the DNA sequences of these genes from the B. methanolicus MGA3 genome, which we found in the NCBI database.
According to the publication by Bogorad et al.[2] the xpk coding sequence for the MCC pathway is derived from the Bifidobacterium adolescentis genome.
To avoid illegal restriction sites and to adapt the coding sequences of mdh, hps, phi and xpk to the codon usage of E. coli, the coding sequences were optimized with the "Optimize Codons" tool of the software "Geneious".
Subsequently, we designed and ordered linear gBlocks with additional extensions up- and downstream the coding sequence. Each of the 58 bp upstream extensions contain an overlap to pSB1C3, the BioBrick prefix and the BBa_B0034 ribosome binding site plus the BioBrick scar TAGTAC. Downstream the CDS, we added an 41 bp extension with the suffix and another overlapping region to pSB1C3.
Since the CDS of the xpk is too long for gBlock synthesis, we devided its sequence into two half and designed extensions for both fragments to assemble them together with a linearized pSB1C3 backbone via CPEC.
Results
mdh, hps and phi were successfully cloned into the pSB1C3 backbone via restriction and ligation. The two xpk fragments and a linear pSB1C3 fragment were assembled in a CPEC reaction. All four resulting BioBricks were sequenced and submitted to the BioBrick registry.
| BioBrick |
construct |
sequencing confirmed plasmid |
confirmed cryo
|
| K1585210 |
RBS.MDH in pSB1C3 |
#9PBD# |
#WNFY#
|
| K1585211 |
RBS.HPS in pSB1C3 |
#H4M9# |
#863A#
|
| K1585212 |
RBS.PHI in pSB1C3 |
#ENKK# |
#AE8V#
|
| K1585213 |
RBS.XPK in pSB1C3 |
#KFEY# |
#8WY9#
|
Laboratory Notebook
References
- ↑ Witthoff S, Schmitz K, Niedenführ S, Nöh K, Noack S, Bott M, Marienhagen J. Metabolic engineering of Corynebacterium glutamicum for methanol metabolism. Appl Environ Microbiol. 2015 Mar;81(6):2215-25. doi: 10.1128/AEM.03110-14. Epub 2015 Jan 16. PubMed PMID: 25595770; PubMed Central PMCID: PMC4345391.
- ↑ Igor W. Bogorada, Chang-Ting Chena, Matthew K. Theisena, Tung-Yun Wua, Alicia R. Schlenza, Albert T. Lama and James C. Liaoa: Building carbon–carbon bonds using a biocatalytic methanol condensation cycle