For our molecular biology department, we finished the interlab study (see here), and were able to create five viable biobricks out of our collection that should include about 11 different biobricks for a siderophore synthesis collection.
In general, we tried to clone 30 kbps, express nine proteins, including two huge synthetases plus characterize two promoter regions and all that to synthesize an optimized version of a small peptide originating from Pseudomonas aeruginosa utilizing none of the native synthetases. It turns out that the cloning of 30 kbps is quite a task even more so when the sequences inherit a way higher GC-content from their origin organism than the host organism is used to.
Therefore, we designed four plasmids with different properties and different inducers. Of which two were designed to express our target siderophores nor-pyochelin (NPch) and yersiniabactin (Ybt), induced by different promoting regions so that we only needed to transform a single strain that would be able to do both if necessary. Our other plasmids were designed to include transportation mechanisms for siderophores for Ybt and Pch. The latter plasmids were designed to create individual testing strains to mimic the potential effects of our product, gallium-siderophores, on Pseudomonas and Klebsiella species in E. coli.
So essentially, we wanted to alter the non-ribosomal synthesis pathway of the catecholate siderophore yersiniabactin to create nor-pyochelin an improved version of one of Pseudomonas aeruginosa siderophores, to eventually utilize them as trojan horses against its organism of origin. A big upside of the experiments we planned was that we could have synthesized two different siderophores in a single organism simply by combining two different substrates to initiate the expression of two different synthetases. Which would have allowed us to test the gallium-siderophore hypothesis vs. two completely different strains that cause pneumonia and both tend to be multiresistant, Klebsiella pneumoniae and Pseudomonas aeruginosa.
What we did achieve: With the support of the Evry iGEM-Team, through a Golden Gate Assembly, we could realise the cloning of one of our big synthetases, HMWP2, and could validate it by gel analysis and sequencing. Thus, we finished the basic part and no are able to precede more efficient. The same applies to the genes dhbE, sfp, and tired, genes we got delivered as basic parts and got a promotor cloned. In addition, we got tired cloned with a terminator and hence can use it in one of the completed biobricks.
Also, a characterisation for tired was realised by experimenting BBa_J04500::tired in E.coli BL21(DE3) cells and inducing it with a OD[595 nm]=0,4 with IPGT, and with IPTG and 10 µg/mL L-Cystein, measuring the kinetic after two hours.
As it can be derived from the growth curve, the in addition to the IPTG with L-Cysteine induced bacteria has a higher metabolic load.
Beside the measurement of the OD with 595 nm a kinetic was additionally measured after the induction at 200 nm to 800 nm.
Thiazolinyl imide reductase, is a protein and is a common but unique catalysator in NRS for peptides including cyclized cysteine, which this reductase forms out of linear cysteine creatin a thiazolin ring.
To do so cysteine needs to be bound to a a C-donator by its amino group. Without a C-Atom to connect cysteine cannot cyclize.
Which explains why a low peak was observable while expressing it in cells were structures, as intermediates, similar to our target siderophores (Pyochelin, Yersiniabactin) might be formed. While we could not detect a shift in fluorescence in cell debris with tired in dissolved in the media.
Since the measurement data is still quite inconclusive this needs further investigation.
In addition to tired we also completed the promoter PchR. To characterise it, the promoter previous to GFP (BBa_E0240) was cloned and the fluorescencintensity was ascertainedto draw conclusions to the activity of the promotor.
The Pyochelin-Complex Regulated Promotor (PchR) is the native pyochelin promotor of Pseudomonas aeruginosa (here: Pseudomonas aeruginosa PAO1) that is activated by its own product complexed with metal-ions, the native pyochelin-iron complex.
The Pyochelin-Complex Regulated Promotor is most likely a leaky promoter that induces the Pyochelin synthesis if the Pyochelin-iron3+-complex binds to the receptor. Most siderophore-receptor-complexes need to be leaky since the siderophore production is only necessary under certain circumstances and self-induced. In this context, the leakiness is not to be seen as a flaw but a feature, including a biosensor for iron3+-ions in the expression system that makes sure that no unnecessary metabolic load is exhibited. Therefore, the iron acquisition method is highly efficient and only expressed when the circumstances demand it.
Our goal was to characterize the promoter and measure if pyochelin-gallium3+ and Nor-pyochelin-gallium3+ complexes induce the native promoter to estimate the metabolic excess overload that our antibiotic-compound induces on native Pseudomonas aeruginosa.
To characterize the promotor, we cloned the 960 bp long DNA part before GFP. As a template, we used the BioBrick BBa_E0240. This is a GFP-tri-part consisting of the ribosome binding site BBa_B0032, GFP with the number BBa_E0040 and the double terminator BBa_B0010 und BBa_B0012.
We transformed the plasmid pSB1C3::PchR::BBa_E0240 in Escherichia coli DH5a cells. For the expression tests the cells were cultivated to an OD [595 nm]= 0,372. Then a 96-well plate (black plate with transparent ground) was loaded with the cells and different concentrations of NPch-Fe-, NPch-Ga-, Pch-Fe- und Pch-Ga-complexed siderophores. Loading scheme:
Over the next eight hours the plate was incubated at 37 °C and 220 rpm and we measured the absorption at 595 nm and the fluorescence of the GFP, (excitation wavelenght = 475 nm and emission wavelength = 509 nm) in a plate reader every 18 min.
Growth and fluorescence of the cells induced with NPch-Fe-complex over the entire time:
Growth and fluorescence of the cells induced with NPch-Ga-complex over the entire time:
Growth and fluorescence of the cells induced with Pch-Fe-complex over the entire time:
Growth and fluorescence of the cells induced with Pch-Ga-complex over the entire time:
The growth curves for the cells induced with the siderophore-gallium-complex show an almost periodically drop of the absorption. Therefore, the assumption is that the induction led to a periodic cell death. The periodic cell death explains the fluctuation of the absorption. We suppose that the cells take up the gallium-siderophore-complex and die due to the sudden release of the cytotoxic gallium.
The fluorescence was divided though the absorption to get a relation of the fluorescence and the cell growth. To remove the leakiness out of the equation we only measured the cells that were transformed with the plasmid but not induced with siderophores (called “blank” in the following).
Fluorescence against absorption of the cells induced with the NPch-Fe-complex.
The graph shows that all values for a NPche-Fe-complex concentration of 0.195 µM bis 50 µM lie above the blank value and prove therefore an induction of the promotor. Lower concentrations induce weaker than the higher concentrations.
Fluorescence against absorption of the cells induced with the NPch-Ga-complex.
The graph shows an induction of the promoter as all values for the NPch-Ga-concentrations of 1.563 µM to 50 µM lie above the blank value. Lower concentrations induce weaker than higher concentrations.
Fluorescence against absorption of the cells induced with the Pch-Fe-complex.
The graph shows an induction of the promotor as the values for all Pch-Fe-complex concentrations between 6.25 µM and 50 µM lie above the blank value. Lower concentrations induce weaker than higher concentrations.
Fluorescence against absorption of the cells induced with the Pch-Ga-complex.
The graph shows that only the values for the Pch-Ga-complex concentration of 50 µM lie above the blank value and induce the promotor. Lower concentrations induce weaker than higher concentrations.
The graphs of the fluorescence against the absorption show a more effective induction of the promotor for the NPch-siderophore-complexes than for the Pch-siderophore-complexes. Also, the iron-complexed siderophores provoke a stronger induction than the gallium-complexed siderophores, as the for the induction needed iron-siderophore-complex concentrations of 12.5 µM, 25 µM und 50 µM lie above the gallium-siderophore-complex concentration of 50 µM. The relevance of the cell death could not be further quantified. The NPch-siderophor is more effective than the Pch-siderophore as the signals of the corresponding concentrations are significantly stronger for the NPch-siderophore than the Pch-siderophore.
Furthermore, the receptor must be leaky as even the non-induced cells showed a fluorescence that is partly even slightly higher or almost equal to values for cells induced with a low concentration of a siderophor-complex. Also, the graphs show that there seems to be a saturation of the promotor, as the curve flattens after a certain time. This can be attributed to the fact that there is only a certain number of receptors that is depending on the number of cells and therefore limits the GFP production and consequently sets the intensity of the fluorescence.
In general, we lacked the time to troubleshoot PCRs, ligations and transformations, which definitely hamstrung our end results. However, there will be more research regarding siderophore therapies taking place, now that we established the necessary technologies at the University of Hamburg.
To analyse the effect of siderophore-Ga-complexes on P.aeruginosa several MIC-assays (Minimal Inhibitory Concentration) were conducted. A final MIC could not be determined due to the problem that the right media to perform the tests were lacking.
The toxicological effect of siderophores on Cystic Fibrosis Bronchial Epithelial (CFBE) cells was investigated with an MTT-test, which measures the vitality of the cells through their metabolism.
It can be said that the toxic effect appears due to the alcohol the siderophores are dissolved and due to the siderophores themselves.
Full version: see here
The products pyochelin, NH-nor-pyochelin and 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid were synthetized.
The presence and purity of the products NH-nor-pyochelin and 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid was confirmed with 1H-, 13C- and 2D NMR spectroscopy while the presence of pyochelin was confirmed using only 1H-NMR, since 2D spectra were not available at that point. The pyochelin was contaimnated to a certain degree, but it was nevertheless used for subsequent testings. The amount of pyochelin was not sufficient for purification.
UV-Vis spectra of Pyochelin, NH-nor-pyochelin and 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid were then recorded. To investigate complex formation with iron(III) and gallium (III), solutions of salts including both metals (gallium(III) nitrate and iron(III) chloride) were mixed with the siderophore solutions. UV-Vis spectra were recorded to investigate the kinetics of the complex formations (see here). Finally, siderophore-gallium complexes were created and used for toxicity tests.
Sadly our first charge of microfluidic devices broke down when we tried to coat them with an ECM-solution to grow our cells on. And we were not able to produce a second charge before wiki freeze.