This part is composed of 3 subparts :
This DNA sequence codes a Lysine decarboxylase (Streptomyces coelicolor) which is an enzyme from the lyase family that converts lysine into cadaverine. The enzyme releases the carbonyl group of the lysin amino acid. Cadaverine (or 1,5-diaminopentane) is a primary diamine which renders the medium alkaline. The lysine decarboxylase is an enzyme whose synthesis is promoted by anaerobiosis and an acidic pH. This enzyme is also the first step in the production of desferrioxame B which is a siderophore.
We succeed to produce DesA under an Arabinose indcution.
We registered the original sequence of this subpart in the iGEM registry of standard parts (BBa_K1951000). We optimized our sequence for E. coli and ordered the synthesis by addition of an inducible promoter.
As you can see in the registry all our expectations on this BioBrick were validated and it worked fine.
The purpose of this biobrick is to produce desferrioxamine B siderophore by a controled manner, for controlled potential toxicity. This is also to reconstruct the full pathway of this siderophore production.
This biobrick was made from 6 parts:
Available in the registry :
Designed by our team :
Siderophores are small, high-affinity iron chelating compounds secreted by microorganisms such as bacteria, fungi and grasses. Siderophores are amongst the strongest soluble Fe3+ binding agents known.
Previous work showed that siderophores are able to catch other metals by default. Especifically, many articles showed a high affinity of Desferrioxamine B (produce by Streptomyces coelicolor) for tetravalent metal ions like platinum. These results encouraged us to make a biobrick coding the sequence corresponding to the 4 enzymes involved on the metabolic pathway of Desferrioxamine B. E. coli was used to produce this biobrick. Produce a gram positive bacteria pathway in a gram negative bacteria is restrictive  , considering the risk of toxicity for themselves. To counter this potential issue, we regulate transcription using the control of an inductible promotor (pBAD/araC).
Desferrioxamine B is a really well referenced siderophore.
Deferoxamine acts by binding free iron in the bloodstream and enhancing its elimination in the urine. By removing excess iron, the agent reduces the damage done to various organs and tissues, such as the liver. Also, it speeds healing of nerve damage (and minimizes the extent of recent nerve trauma).
We investigated if the DesA, DesB, DesC and DesD proteins were well produced by our biobrick BBa_K1951011 using SDS PAGE.
To do this we performed SDS PAGE (protocol) and stained with coomassie blue using cells containing this biobrick. From an over night starter, cells were diluted and grown from Abs(600nm)=0.2 to Abs(600nm)=1. Then 1UOD of cells (1.67ml at 0.6OD) was collected and centrifuged at 5000g for 5min. After removal of the supernatant, the cell pellet was resuspended in 50µL SDS-PAGE sample buffer. We heated the mix at 95°C during 15min. The sample was loaded onto a polyacrylamide gel and migrated during 50min at 180V. Staining was done using coomassie blue.
We compared the production of proteins in different background :
- E. coli TG1 strain with pSB1C3 containing the RFP coding sequence (negative control)
- E. coli TG1 strain with des operon (BBa_K1951011) before and after induction. (You can observe the production of the 4 proteins on the figure on the right; left : before induction, right : after induction)
Results showed the production of the 4 proteins DesA, DesB, DesC and DesD, all involved in the desferrioxamine B biosynthesis pathway. We can notice that a leak of the promoter pBAD, indeed we note the production of Des proteins without arabinose induction.
All the functional tests and experiments, show this part is functional, are listed here.
This part is a composite part composed of 2 Biobricks :
Flagellin C (FliC) protein from Escherichia coli strain is the main protein constitutive of the flagelar filament and is involved to promote bacterial swimming. This sequence is conserved in many bacterial strains. It has been demonstrated that flagellin has the ability to adsorb precious metal such as platinum or gold. We made a FliC mutant by transduction using phage P1 in a E. Coli W3110 strain. Then we have complemented the fliC mutant W3110 with BBa_K1951008 and performed a swimming test for every background. The result has shown that swimming was recovered into the complemented fliC mutant W3110
We constructed a fliC deletion mutant that is unable to swim, from the wild-type strain W3110. The ability to swim was restored to this mutant by our biobrick, as can be seen in the illustration here. This demonstrated that the protein can be correctly inserted into flagella and functions.
The subparts were assembled using standard BioBrick Assembly.
Flagellin (FliC) protein from Desulfovibrio vulgaris strain is the main protein constitutive of the flagellum filament and is involved in bacterial swimming. This protein is conserved in many bacterial strains as the capacity of swimming given by the flagellum confers a great selective advantage.
The propensity of the immune response to flagellin may be explained by two facts:
CsgA is the major structural subunit of the curli fimbriae. Curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation. Curli also mediate host cell adhesion and invasion, and they are potent inducers of the host inflammatory response. The biobrick contains a strong promotor.