4.1 BioBricks and part collections

Although this year iGEM Berlin participated for the first time in the iGEM competition, we succesfully constructed, sequenced and submitted 15 biobricks.
In the following table you can see all of our constructed biobricks
<groupparts>iGEM014 Berlin</groupparts>
Next to standardized parts like BBa_K1438000, we decided to sent in expression devices meaning that our ferritins are on a plasmid which can be expressed. We decided to use the pQE_80L expression vector as a standart expression vector as we did not receive the distribution in time and a real standard expression vector is seems to be missing in the registry. pQE80_L is an standard expression vector that was provided by AK Budisa. It has an N-terminal His-tag for protein purification, inducable with IPTG and has an T5 promotor for efficient expression. As we noticed that expressing our ferritins in this vector works better than in other ones, we decided to submit our parts in this plasmid. We also submitted the used vector as a part to the registry.

This allowed us to construct and submit the iGEM Berlin Ferritin library. A part collection that includes six different ferritin proteins with different properties on standard expression vector. In the following table our favourite ferritin expression devices are shown.

TODO TABLE_Ferritin



As an alternative strategy to ferritins, we constructed several expression devices for metallothioneins and phytochelatin synthases in different systems. A.) A two plasmid system where PPMT and ATPCS are expressed on different vectors. B.) A one plasmid system where PPMT and ATPCS are co-expressed C.) An ATPCS and PPMT fusion protein expression system.

Considering these designs we constructed a variaty of different ATPCS and PPMT expression devices as shown in the following table

TODO TABLE_PPMTATPCS

For calculation of the molecular masses ProtParam from Expasy was used.

4.2 SDS Page of ferritin expression devices

We performed SDS PAGE before and prior induction of our cells and noticed that most parts are expressed well. A few SDS PAGE samples are shown in the following. Please find the corresponding molecular weights in the upper table.

TODO SDS_BFR

TODO SDS_FTNA2

TODO Hu_ferritin

TODO JBFS_mil_ferritin



4.3 Iron Sensitivity Assay

We tested various iron precursor substances like FeSO4 , Fe-citrate, FeCl2, Fe-gluconate and Fe-ascorbate resulting as Fe-citrate as the most promising because of it beeing more soluble in water then others.

Following the hypohesis that we are looking for a chassi that takes up more iron then others and therefore will be more sensitive to high iron concentrations. We screened different E. coli wildtyp strains.

TODO tolerance_wt tolernace assaay.png The first assay resulted in MG1655 and RV308 growing on 0.5 mM iron citrate whereas E. coli Nissle and DH10b arenot able to grow at 0.5 mM iron. RV308 is able to grow even up to 2 mM iron citrate. This result was really surprising as most papers we were refering to were working with iron citrate concentrations 1mM and more. This makes us wonder how they were able to obtain any results using strains like MG1655 and DH10b.

4.4. Iron Uptake Assay
To quantify the iron uptake of E. coli we conducted the prussian blue assay also called berlin blue assay using Iron(II,III) hexacyanoferrate. We screened our ferritin expression devices as well as different knockout strains for increased iron uptake. Our working hyptothesis was that expression of ferritin proteins increases the iron capacity inside of the cell and because of this E. coli would incorporate more iron.

An increased iron uptake not only proofs that ferritin proteins are functional inside of the cell but also that more iron for magnetizing E. coli is available inside the cell.

TODO Bild Assay

4.5. Fluorescence and confocal microscopy and magnetization test

The moment of truth came when we analysed our cells that expressed our biobricks and after were incubated in an iron rich solution inside of a microcapillary with a confocal microscope or even sometimes with an flourescence microscope.

We created an playlist on youtube with our results- so you can watch our samples under different conditions showing magnetism or not. Video of magnetising tests

We were excited to see that under different ion concentration magnetic movement was noticable. Our test involved three stages(black screen indicated change between stages).

1. Capillary with cell suspension without neodym magnet
2. Capllary with cell suspension with magnet from the lower picture side
3. Capllary with cell suspension without neodym magnet
4. Capllary with cell suspension with magnet from the upper picture side


We were really excited to see moving parts under the microscope and cells collecting on the microfluidic channel. However soon we noticed that the moving bits we were analysing under the microscope might be infact precipitated iron salts. Therefore, we conducted flourescence microscopy to validate our experiments. (data not shown)