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

Next to standardized parts like BBa_K1438000, we decided to send 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 standard 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, inducible with IPTG and has an T5 promoter 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.


Table of constructed biobricks for expression of the iGEM Berlin ferritin library

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 variety of different ATPCS and PPMT expression devices as shown in the following table



Table of constructed biobricks for expression of the alternative metal binding proteins library

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.


15% SDS gel showing the expression of BFR in E. coli DH10b


15% SDS gel showing the expression of FtnA1 in E. coli DH10b


15% SDS gel showing the expression of HuFerritin in E. coli DH10b


15% SDS gel showing the expression of jbfs_mild_Ferritin in E. coli DH10b

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 being more soluble in water than others.

Following the hypothesis 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 wild type strains.



As you can see in the picture above the first assay resulted in MG1655 and RV308 growing on 0.5 mM iron citrate whereas E. coli Nissle and DH10b are not 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 hypothesis 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.


Iron uptake assay for our constructed ferritin expression devices as well as a fief knockout strain. As a control a GFP expressing culture and a wild type strain without knockout (BV125) were analysed as well. All ferritin expression devices show significantly that iron was better incorporated as in the GFP expressing control.


4.5. Fluorescence and confocal microscopy and magnetization test



The moment of truth came when we analyzed 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 a fluorescence microscope.

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

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

1. Capillary with cell suspension without neodym magnet
2. Capillary with cell suspension with magnet from the lower picture side
3. Capillary with cell suspension without neodym magnet
4. Capillary 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 analyzing under the microscope might be in fact precipitated iron salts. Therefore, we conducted fluorescence microscopy to validate our experiments. (data not shown)