Our project uses a total of seven enzymes from the bacterium Formosa agariphila. There are three degradation enzymes and three sulfatases. For the sulfatases to work, an additional enzyme is used – the formylglycine-generating enzyme (FGE). This one, according to Franck Daligault, modifies the sulfatase catalytic site in a co-translational way, by transforming a serine or a cysteine into formylglycine, thus activating the sulfatases.
E.coli BL21 (DE3) strain
The plasmids are designed according to the Gibson Cloning method (details below). We produce our enzymes in an E. Coli BL21 (DE3) chassis. This strain has the gene encoding for T7 RNA polymerase in its genome. That is why we also use a T7 promoter and terminator.
Plasmid
We use two types of plasmids based on those described by Reisky and al. (2019). Indeed, in this article, they used pET-type plasmids.
pET 11 plasmid
This plasmid is used for the expression of the three so-called degradation enzymes (P31_GH39, P10_Plnc, P30_PL28) and the three sulfatases (P36_S1_25, P18_S1_7, P32_S1_8).
Fig. 1 each pET11a plasmid is containing one of the following enzymes: degradations enzymes P30_PL28, P10_Plnc, and P31_GH39 and sulfatases P18_S1_7, P32_S1_8, and P36_S1_25.
Plasmid elements
T7 Promoter and Terminator
LacO operator sequence
LacI protein sequence : inhibitor protein fixing on lacO
Ampicillin resistance gene
Gene of interest containing our enzyme’s ORF, base pairs allowing Gibson Cloning and a 6xHis Tag for protein purification.
Mechanism IPTG is a non-hydrolyzable lactose analog. In the absence of IPTG, the LacI protein is produced and tetramerized. The LacI tetrameric complex binds to the LacO operator sequence which blocks the transcription by T7 DNA polymerase.
The addition of IPTG to the culture medium induces the transcription of our genes of interest. As a matter of fact, IPTG complexes with the inhibitory protein LacI, preventing it from binding to the operator sequence. The promoter is therefore accessible for the polymerase and the transcription can actually take place.
pEVOL-1 plasmid
This plasmid is used for the expression of the FGE. Our supervisor, Dr. Emilie Camberlein, gave us this plasmid because it contains an arabinose inducible promoter, which is convenient for our experiments.
Both the FGE and sulfatase plasmids are co-transformed.
Fig. 2 pEVOL-1 plasmid containing FGE.
Plasmid elements
Arabinose Promoter
Sequence coding the protein araC : Inhibitor protein fixing on Ara promoter
Chloramphenicol resistance gene
Gene of interest containing our ORF for the FGE, bases pairs allowing Gibson
Cloning and a 6xHis Tag for the protein purification.
Mechanism
In the absence of arabinose, the AraC protein, which has two DNA binding sites, is produced. The AraC protein binds to the Ara Promoter which will be blocked and will bind the DNA. When adding arabinose to the culture medium, it binds to the AraC protein which then becomes an activator protein. Thus, transcription is possible and our protein is produced.
Gene of interest
The ORFs of our 7 genes of interest are retrieved from NCBI. We chose the Gibson cloning technique, which allows genes to be cloned without the use or restriction sites for enzymes.
Gibson cloning
Gibson cloning is a one-tube reaction (containing 3 different enzymatic activities in a single buffer: exonuclease, DNA polymerase, DNA ligase). The software SnapGene is used to design our plasmids. Our enzyme ORFs are ordered on “custom gene synthesis” from our sponsor IDT. On both sides of our ORFs, we added 20 base pairs. Therefore, we are only using 8 primers, 4 of them are used for the pET Plasmids and the other 4 for the pEVOL plasmids.
Figure : 4 primers (in purple) at the end and at the beginning of the gene are hybridized on the base pairs added for Gibson Cloning (in grey) on both sides of the ORF (here, it is the sequence coding for the FGE protein, shown in blue). On the left, the figure corresponds to the beginning of the FGE’s ORF and on the right, it corresponds to the end of the sequence. Designed with Snapgene.
Thus, each ORF is in a cloning vector. During the process of Gibson assembly, our primers hybridize on both sides of our ORF. This allows us to clone our ORF from our parent vector to our final vector - pET or pEVOL in the right orientation in relation to the promoter and terminator.
Purification
Finally, the His-Tag sequence added in C-terminal of our ORF will allow us to purify the enzymes once they are produced, by using an immobilized metal affinity chromatography (IMAC) with nickel resin.
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