Team:Chiba/Project/store

Storage

1.Introduction

    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.
    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。
    These two effects enable isolation of iron in ferritin and enhance E.coli iron tolerance.

     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.

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 strong RBS and BBa_K1057009 with middle 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).

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。

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

2.4.磁性評価

3.Results & Discussion

3.1.プラスミド

3.2鉄耐性の評価

3.3磁性評価

4.Conclusion

それは・・