"
Team:UEA-JIC Norwich/Nittygritty-moss
From 2011.igem.org
Moss
We have decided to work with both Physcomitrella patens and algae (Chlamydomonas reinhardtii) with the intention of encouraging their use in iGEM. Physcomiterella patens itself has benefits for use in iGEM. It's a model organism which can be transformed with inexpensive, widely available equipment using PEG (poly ethylene glycol) mediated methods, and provided a thorough project and experimental plan is constructed early it can be transformed and grown within the ten week time period of a typical iGEM project. The full genome has been sequenced and there is a lot of existing data to support iGEM teams who wish to work with it.
A significant benefit to moss is that its transcription pathways are well conserved and it doesn’t suffer from codon bias, in theory providing a greater success rate in translation and synthesis from genes that have been derived from other organisms. If this is found to be correct, work with moss could avoid costly and time consuming gene optimisation and synthesis, which we believe is currently seen as a significant restriction on plant work in iGEM.
Physcomitrella fact file:
Name: Physcomitrella patens
Attributes:
No codon bias
Eukaryotic photosynthetic organism – therefore its post translational modifications will more closely reflect those seen in plants and other higher organisms when compared to, for example, E.coli
Genome is fully sequenced, well documented growth methods and transformation protocols
No genomic integration of plasmid
Difficulties of Use:
Low transformation frequency (though higher than Chlamydomonas)
Takes around a month to grow
Growth Conditions:
Requires Knop’s media
Must be grown at 37°C
Must be grown in sunlight (in light for 16 hours, darkness for 8 hours)
Recipe for Knop's media:
1.84 mM KH2PO4
3.4 mM Ca(NO3)2
1 mM MgSO4
2.72 mM Diammonium tartrate
54 μM FeSO4.7H2O
9.93 μM H3BO3
1.97 μM MnCl2.4H2O
0.23 μM CoCl2.6H2O
0.19 μM ZnSO4. 7H2O
0.22 μM CuSO4. 5H2O
0.10 μM Na2MoO4.2H2O
0.168 μM KI
0.8% agar for solid medium
6% mannitol
All of the above ingredients have been autoclaved, then 10 mL of 1 M CaCl2 (autoclaved) has been added before pouring the plates.
The lack of a codon bias means that P. patens should be able to express genes from all organisms equally well, with no optimal preference for the specific codons used to code for any amino acid, and a similar lack of preference for a specific stop codon. The ability of P. patens to retain plasmids episomally is also an advantage. This lack of genome integration gives it a higher transformation frequency in comparison to algal species.
The lengthy growing time of P. patens is a major disadvantage, but if properly planned the impact of this on projects can be minimised. Once transformed, the growth of colonies after around a week serves as proof of the uptake of a given plasmid. From the time colonies are first observed relevant tests and experiments can be conducted in accordance with the Biobrick transformed into the moss. In this way, it is not necessarily vital to wait a month for the full growth.
Moss Plasmid Design:
Below is the plasmid designed for use as a moss/ algae specific vector, showing an example of a composite:
