Difference between revisions of "Team:Nantes/Description/design"
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Revision as of 02:58, 17 December 2021
Design
Our project is using a total of seven enzymes from the bacterium Formosa agariphila.
There are three so-called degradation enzymes and three sulfatases.
In order for the sulfatases to work, an additional enzyme is used : Formylglycine-generating enzyme (FGE).
Why this enzymes ?
The ulvan degradation cascade described by Reisky et al. (2019) shows us that the degradation enzymes P30_PL28, P10_Plnc and P31_GH39 have a significant effect on ulvan.
Based on this research, it’s why our team decided to use those three enzymes.
Regarding the sulfatases, we based our decision on the same article. The P18_S1_7, P32_S1_8 and P36_S1_25 show an activity more or less important on ulvan.
The strain E.coli BL21 DE3 was chosen for the expression of our enzymes.
Plasmid selection
pET11a
This plasmid is used for the expression of the three so-called degradation enzymes and the three sulfatases.
pEVOL-1
This plasmid is used for the expression of the FGE.
FGE and sulfatases are either co-expressed or put together once produced.
The His-Tag sequence allows us to purify the enzymes once they are produced, by using an immobilized metal affinity chromatography (IMAC) with nickel resin
Problems encountered
pET11a - pEVol-1
We were unable to amplify it by PCR.
For pET11a we can use another method to get it, but for pEVOL-1, the problem is that it’s impossible to do the digestion protocol because there are no easy restriction sites on the plasmid.
Modifications made
pET11a
We have redesigned the reward and forward primers.
This modification did not allow the expression of the plasmid.
We believe that restriction sites should be added to the insert sequences to perform a restriction + ligation technique
pBAD_crtEBI_19 (modified)
pBad-crtBI_19 could be suitable.
We just need to change the ori for a p15 ori which is compatible with the pET11.
We also need to make a new design for FGE insert to be compatible with the new plasmid.