A Gene-Protein-Reaction Association parsing script for SBML models
In SBML version 2, level 3 Flux Balance Constraints(“fbc”) package models,
Gene-Protein-Reaction Associations (GPR associations) are being stored under <GeneProductAssociation>
objects, presented in a binary tree structures branching from boolean operators.
One reaction could have multiple possible GPR associations, in that case, the information
under <GeneProductAssociation> object would be complex and the correct combinations of
geneProducts is not directly obtainable. Thereby on the path of building Forbidden FRUIT,
Mingdong has built a GPR associations parsing method with using a python API library
Libsbml to handle the file. This method is
able to go through a SBML model and extract all the possible GPR associations for all
example script is available on Github for other users of SBML models to use.
We were able to contribute knowledge to future Dutch teams through our consumer survey, which explored the perception of genetic modification among students in Amsterdam. This survey was empirically designed and executed to ensure scientific validity. We had nearly 150 participants in our target group complete our survey, sufficient for statistically significant results, which can be viewed here
What did we use?
We used the Anderson promoter collection which resulted from a library screen by Chris
Anderson. The group
of Vaseduvan recently published a paper comparing the strength of these and other promoters
in Synechocystis PCC6803 (Synechocystis) and UTEX 2973 (Vaseduvan, et
al. 2019). We used this relation in the design of our modification strategies in Synechocystis.
Based on their strength, we choose the following promoters: Part: BBa_J23104
and Part: BBa_J23119.
How did we use it?
We aimed to express a gene
neighbouring the pyk2 gene which we knocked out in Synechocystis to ensure the
transcription of this gene is not disrupted by our gene knockout. The strength of the promoter
natively driving this gene, sll1276, is unknown. Since there are strong suggestions that
the sll1276 gene is essential, we choose a promoter of medium strength and one of a high
strength (Yao, et al. 2019). According to the study of Vaseduvan, et al (2019)
in a medium expression, while Part:BBa_J23119 resulted in a
high expression in Synechocystis. We made two DNA templates using the different
promoters. The DNA templates were used in a CRISPR-Cpf1 experiment to knockout pyk2.
Replacement of pyk1 with irp9 in Synechocystis: Part:BBa_J23119
A problem we faced when implementing the strategy to produce salicylic acid with Synechocystis
is that the cells did not survive when all genes which are related to the production of the
coproduct (pyruvate) are knocked out. This might be due to the strength of the promoter
driving irp9 expression in the plasmid which provides the production pathway.
Therefore, we tried to introduce an additional copy of irp9 on the genomic locus of
pyk1. To ensure this gene is highly expressed we used the strong Part:BBa_J23119
promoter to drive its expression.
How did we use it?
To help other teams to choose a suitable promoter in Synechocystis PCC6803 and UTEX
2973 we added the information we found in the paper from Vaseduvan (2019) to the
Registry main page of the Part:BBa_J23119 part.
Vasudevan, R., Gale, G. A. R., Schiavon, A. A., Puzorjov, A., Malin, J., Gillespie, M. D., Vavitsas, K., Zulkower, V., Wang, B., Howe, C. J., Lea-Smith, D. J., & McCormick, A. J. (2019). Cyanogate: A modular cloning suite for engineering cyanobacteria based on the plant moclo syntax. Plant Physiology, 180(1), 39–55.
Yao, L., Shabestary, K., Björk, S. M., Asplund-Samuelsson, J., Joensson, H. N., Jahn, M., & Hudson, E. P. (2019). Pooled CRISPRi screening of the cyanobacterium Synechocystis sp. PCC 6803 for enhanced growth, tolerance, and chemical production. BioRxiv, 2020, 823534.