Team:Chiba/Project/store

Sequestration: Fe-storage machine

1.Introduction

    

Fe must be isolated: In order to magnetize E. coli, we need to stuff as much Fe ions as possible in E. coli. Fe(II) could cause Fenton reaction in response to hydrogen peroxide, harmful hydroxyl radicals (OH•). Dilemma is that, feeding too much Fe into cell would kills the host cell. We decided to over express the ferritins that capture and store Fe irons.

Fe container machinery: Ferritin particles are made of two small protein subunits (heavy chain (FTH) and light chain (FTL)) (Fig. 1). When expressed, the ferritin subunits automatically assemble into 24-membered protein cages.

Fig. 1 Complex structure of ferritin

    Heavy chain catalyzes oxidation of iron and stimulate 2Fe(II)＋O2→[Fe(III)-O-O-Fe(III)] reaction

2Fe₂+O₂+(H₂O)x+3→Fe₂O₂(H₂O)x+4H+H₂O₂

    On the other hand, light chain takes up Fe(III). By functional expression of these two polypeptides, virtual concentration of Fe inside cell can be much reduced. Interestingly, the storage capacity, the complex size, and FTH/FTL ratio can vary from species to species. Generally speaking, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions.

Choice of ferritin: Because the storage capacity of E. coli ferritin is far less than that of mammarian type, we decided to make BioBrick for the functional expression of human ferritin in E. coli.

Hypothesis: By storing Fe in isolation, the maximum capacity for Fe storage should be elevated. Also, the apparent iron tolerance of E. coli should be also elevated.

2.Experiments

2.1.BioBrick construction

    The ratio of FTH/FTL can be flexible in ferritin complex, and there exist a best composition that gives the highest Fe-storage activity. In heterologous expression of ferritin, the translational efficiency can be fine-tuned so that we could achieve that best composition. So, we constructed BioBricks for the functional expression of human ferritin complex in two formats;
BBa_K1057002: 'middle' RBS assigned for FTH
BBa_K1057009: 'strong' RBS assigned for FTH
In both construct, two ferritin genes (FTH and FTL) are placed under pBAD promoter to control the timing and expression level of these genes. To facilitate this construction process, we modified an existing BioBrick (BBa_I74608 deposited by iGEM 2007 team Cambridge) into the new BioBrick(BBa_K1057012). This enabled us the rapid, in-parallel, and one-pot digestion/ ligation using Golden gate method.

Fig. 2 Cloning procedure of ferritin-producing BioBrick

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2.2. Confirmation of Ferritin Expression

Our new BioBricks BBa_K1057002 and BBa_K1057009 were transfected into in BL21 strain. The expression level of each individual components (H-chain and L-chain) was checked by SDS-PAGE/ coomassie blue. As a control, we also conducted the same experiment with “sfgfp generator”.
The results are shown in Fig. 3. Both in total protein fraction (gel in left) and soluble fraction(gel in right), there observed two characteristic bands: one was with the size of 20 kDa (corresonding to FTH), while the other was 19kDa (corresponding to FTL). These bands were not detectable for the control sample (Lane 3) expressing the control gene sfGFP under the control of pBAD promoter.

Fig. 3 Expression of ferritin in E. coli treated with arabinose.
lane 1. pBAD/araC-ferritin-strong(BBa_K1057009)
lane 2. pBAD/araC-ferritin-mid(BBa_K1057002)
lane 3. BBa_I746908(sfgfp)

2.3.Evaluation of iron tolerance

Experiment: E. coli strain BL21 and SHuffle® were transformed with above plasmids. Here, Human ferritin genes (fth1 and ftl) are under the control of pBAD promoter. To express Human ferritin proteins, arabinose (final conc. 0.2%) was added into media. These ferritin generating cell (10^7 cells) inoculated in to fresh media including various concentration of iron citrate (Fe(III)) or iron ascorbate (Fe(II)). After 12h of incubation (at 37ºC), the final cell density and colony forming efficiency was evaluated. As a control, we conducted the same experiment with the cell harboring BBa_I746908 (arabinose-induced sfGFP generator). For the detail of the iron torelance protocol, see Here.

2.4.Evaluation of magnetism

Now, we finally examined whether BL21 overexpressing ferritin is well attracted to the magnet. Our experimental setup is shown below (Fig. 4).
The details of this experimental protocol is given here.

Fig. 4 Experimental setup

3.Results & Discussion

3.1.plasmid

3.2 Assessment of iron tolerance

Result. 1

Fig. 5 Iron tolerance of E. coli harboring ferritin-expressing BioBricks

•Over-expression of ferritin didn’t affect the growth so much.
•The cell not expressing overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is > 6-8 mM. Virtually, no growth was observed ost cells (both BL21 and SHuffle®) above that concentration.
•The cell expressing human ferritins showed significantly higher tolerance to the ferrous ascorbate.
•We observed significant difference in viability profiles of the two strains BL21 and SHuffle®. In the case of BL21, expression of ferritins improved the cell growth at semi-inhibitory concentration (5-7 mM) of ferrous ascorbate (up to 10 x), but above that concentration, ferritin expression did not rescue the growth of the cell. In case of SHuffle® strain, the final cell density wasn't that elevated, but it rescued the cell growth at higher concentration (7-8 mM) of ferrous ascorbate. We do not know where this difference comes from. It should be noted that SHuffle® is specifically engineered strain so that the cytosolic environment is more oxidizing than the normal E. coli cells.
•The resultant culture was stamped on the fresh agar plate to form colonies. As is shown below (Fig. 5), The higher tolerance of cells expressing ferritin was again obvious: the cell expressing ferritins retains higher viability (colony-forming capacity) than the control.

Fig. 6 Colony-forming capability of E. coli with/without ferritin-expressing BioBricks

3.3 Evaluation of magnetism

Result
Our expectation was very simple; if E. coli is well magnetized, they should be attracted by magnets so that we should see the emergence of a pattern- due to the accumulation of the cell near the magnet. Alas!, however, we did not see any trends of E. coli flock forming around magnet: nothing really happened....

Maybe, we should have done the same experiment in SHuffle® strain where the cytosolic environment is engineered to be more oxidizing. With the same level of iron contents, the ratio of Fe (III), the required state for ferromagnetism), can be significantly improved. Also, the level of iron accumulation was not sufficient at all. Next step we are trying is to further increase in the cellular iron contents by combining this ferritin over expression system with the temporal knocking down of Fur and fieF using CRISPRi technique.

Fig. 7 Our first attempt to attract E. coli by magnet.