Team:Chiba/Project/store

Storage

1.Introduction

     In order to magnetize E. coli, we decided to import as much Fe ions as possible in E. coli. So, we focused on ferritin that stored Fe irons. Ferritin forms 24-mer protein composed by heavy chain (FTH) and light chain (FTL) as shown in Figure 1. The H chain oxidizes Fe and L chain stores it. The reaction in H chain is below.
        2Fe2++O2+(H2O)x+3→Fe2O3(H2O)x+4H++H2O2
The different species has the different ratio of FTH to FTL. Generally, the mammalian ferritin has more FTL on the other hand the ferritin derived from bacteria has more FTH. Because we wanted E. coli to store as much Fe as possible, the mammal ferritin is suitable for our object. Therefore, we decided to introduce human ferritin that can be expressed in E.coli.



    したがって、フェリチンの過剰発現によって、 細胞の鉄の含有量は増加するはずである。また，実効的な2価鉄の濃度が減少し、細胞死が起こりにくくなるはずである。

Figure 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. As a result, giving iron to E.coli excessively leads to the death of 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.

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).

Figure 2. Cloning of ferritin

Table 1. RBS score

Parts link

2.2.Expression check

BL21株によってstr, midをそれぞれ発現させた。これによってフェリチンが発現されたかを確認するため、SDS　pageにより評価を行った。 比較対象としてstrまたはmidの代わりにsfgfpをいれたプラスミドを発現したBL21株を用いた。 以下に全画分および可溶性画分のSDS page を示す。

Figure 3. SDS-PAGE patterns of ferritin extracted from pBAD/araC-ferritin-strong, pBAD/araC-ferritin-mid, and BBa_I746908(sfgfp): (1) pBAD/araC-ferritin-strong , (2) ,pBAD/araC-ferritin-mid, (3) BBa_I746908(sfgfp) 全画分、可溶性画分ともに、strong and middleには19kDaと20kDaにバンドが見られる。これらはそれぞれフェリチンのFTHとFTLに対応している。 また、sfgfpの発現株には、30kDa付近にsfgfpのバンドが見られる。 これによってstr、およびmidでフェリチンが発現されたことが分かる。

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. 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.磁性評価

Assay

3.Results & Discussion

3.1.plasmid

3.2 Assessment of iron tolerance

Result. 1

fig.A
鉄培養液中で12時間培養した後の大腸菌の光学密度を測定するとfig.Aになった。
(鉄培養液に植菌した際の細胞光学密度は約0.01 (OD600)に統一した)

・フェリチンの過剰発現発現は増殖に大きな影響を与えなかった。
・フェリチンを過剰発現していないコントロールの場合，アスコルビン酸鉄濃度が6-8 mMのときに，明らかな増殖阻害がみられ，8 mM以上では，大腸菌の生育が全く見られなかった。
・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
・またSHuffle株においても同様の傾向を示した

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


コロニー形成能の変化も，ほぼ同様の傾向を示した

The ability to form colony changed nearly the same.

3.3磁性評価

Our purpose is to give E. coli magnetism.
To confirm whether BL21s to introduce str and mid each have magnetism, we perform experiment shown below.
The details are shown in assay.
If E. colis have magnetism, they approach to magnets!

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・・