Team:Chiba/Project/store

Sequestration: Fe-storage machine

1.Introduction

     In order to magnetize E. coli, we need to stuff as much Fe ions as possible in E. coli. However, too much Fe would kills the host cell. As a result, giving iron to E.coli excessively leads to the death of E.coli.
We decided to over express the ferritins that capture and store Fe irons. Ferritins form 24-membered protein cages composed by two small proteins called heavy chain (FTH) and light chain (FTL) (Fig. 1).
Fig. 1. structure of ferritin

    Fe(II) in E.coli causes Fenton reaction in response to hydrogen peroxide and produce hydroxyl radical (OH･) which is harmful to E.coli.
    Ferritin is an intracellular protein which has a property to store iron. Ferritin has Heavy chain and Light chain. Heavy chain affects the oxidation of iron and stimulate 2Fe(II)＋O₂→[Fe(III)-O-O-Fe(III)] reaction. Light chain takes in Fe(III) in ferritin. Fe₂O₃(H₂O) has paramagnetism.
    These two effects enable isolation of iron in ferritin and enhance E.coli iron tolerance.

The H chain oxidizes Fe, while L chain stores it. The reaction catalyzed by H chain is shown below.
        2Fe₂+O₂+(H₂O)x+3→Fe₂O₂(H₂O)x+4H+H₂O₂
From species to species, the complex size, as well as the FTH/FTL ratio varies. Generally, the mammalian ferritin complex contains more FTL than FTH, while the ferritin complex from bacteria have reversed compositions. Because the storage capacity of E. coli ferritin is far less than that of mammarian type. Therefore, we decided to make BioBrick for the functional expression of human ferritin E.coli.



    Therefore, value of intracellular iron should increase. Also, Cell death may be difficult to occur because of decreasing Fe(ii) concetration.

2.Materials & Methods

2.1.Parts

     The ferritin has FTH and FTL but the ratio is different in the species. However, it may be depend on the expression. So, we constructed two different human ferritin; BBa_K1057002 with middle RBS and BBa_K1057009 with strong RBS. Further more, we placed two ferritin genes (FTH and FTL) under pBAD promoter to control the expression level ant the timing. For these reasons, we constructed a new BioBrick based on BBa_I74608 (deposited by iGEM 2007 team Cambridge).

Fig. 2. Cloning of ferritin

Parts link

2.2.Expression check

pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid were expressed individually in BL21 strain.
We performed SDS-PAGE to check the expression of pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid.
As a control, we conducted the same experiment with “sfgfp generator”.
The results of SDS-PAGE were shown below.

Fig. 3:SDS-PAGE patterns of proteins extracted from E. coli introduced pBAD/araC-ferritin-strong, pBAD/araC-ferritin-mid, or BBa_I746908(sfgfp): (1)pBAD/araC-ferritin-strong, (2)pBAD/araC-ferritin-mid, (3)BBa_I746908(sfgfp). Left is the result of all fraction, and right is soluble fraction. There exist 2 bands around 20 kDa in both pBAD/araC-ferritin-strong and pBAD/araC-ferritin-mid. The band of 20 kDa is FTH. Another band, the band of 19 kDa, is FTL. There does not exist such bands in Lane(3), but exist a different band in 30 kDa. This is a band of sfgfp.

2.3.鉄耐性の評価

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.A

Fig. A 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磁性評価

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・・