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

Parts link

2.2.Expression of Ferritin in protein level

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

Assay
Experiment: We constructed two kinds of plasmids with which RBS scores are different as shown in Fig. 2. E. coli strain BL21 and SHuffle® were transformed with above plasmids. Human ferritin genes (fth1 and ftl) are placed on the high-copy plasmid under the control of BAD promoter. To express Human ferritin proteins, arabinose was added into media(final conc. 0.2%). The resultant “ferritin generators” were cultured in the presence of iron citrate (Fe3+) or iron ascorbate (Fe2+), and checked final cell density and colony forming efficiency. As a control, we conducted the same experiment with “sfgfp generator”.

2.4.Evaluation of magnetism

We examine whether BL21 overexpressing ferritin is attracted to a magnet.
Our experimental setup is shown below(Fig. 3).
The details are shown in assay.
If E. coli have magnetism, the attract to magnets!

3.Results & Discussion

3.1.plasmid

3.2 Assessment of iron tolerance

Result. 1

Fig. 4

Fig. 4 shows the optical density of E. coli which is cultured in the presence of iron for 12h (at 37°C).
(Approximately 10^7 cells inoculated in to fresh media(2 mL, containing iron)in each.)
・Overexpression of ferritin didn’t affect the growth so much.
・The one without overexpression of ferritin, growth inhibition was observed when the concentration of ferrous ascorbate is 6-8mM. And when the concentration of ferrous ascorbate was over 8 mM, the growth of E. coli wasn’t observed at all.

・一方，フェリチンを過剰発現した大腸菌は，アスコルビン酸鉄濃度5-7 mMの範囲では，アスコルビン酸鉄不在下よりも増殖がよく?~ちょっとここ考えます，また9-10 mMのアスコルビン酸鉄濃度においても，細胞は増殖した
・E. coli strain SHuffle® showed same results.

Result. 2


The ability to form colony changed similarly.

3.3 Evaluation of magnetism

Result

Each E. coli don`t move at all.
We consider this cause is uptake quantity of Fe(iii)　is not sufficiently.
So, expression levels of ferritin in each E. coli is not sufficiently.
Future subject is to increase uptake quantity of Fe(iii) to  knock down/out Fur and fieF.

4.Conclusion

それは・・