Team:Chiba/Project/uptake

Uptake

1.Introduction

    Fur (Ferric uptake regulator) controls iron metabolism such as taking iron in or storing iron.
    In most cases, it combines with Fur box (which is near the promoter) and regulates transcription.

    When iron is rich, Fur becomes active and when Fur becomes active, it restricts the expression of iron transporter, and that means that the iron uptake would stop. At the same time, Fur restricts the expression of RyhB. RyhB is one of the sRNA that restricts the expression of Ferritin, so making Fur active leads ferritin to express.
    Conversely, when iron is lacking, Fur becomes inactive, and the iron transporter would work normally, so iron would be taken in. RyhB would also work normally, so expression of ferritin is restricted and Ferritin wouldn't be expressed.

    In short, if we knockdown/knockout Fur, iron transporter would be active so iron would be taken in, but the expression of Ferritin is stopped by RhyB.

    Also, there is a Regulator called fieF (ferric iron efflux). It makes iron and zinc flow out of cell and controls detoxification of cell. When fieF is knocked down/out, the tolerance of cell to iron would be lowered.

    Fec has a character that can recognize ferric citrate and taking Fe(III) in. Fec is controlled by Fur and when the density of Fe(III) in the cell is too high, Fur restricts Fec and stops iron uptake.
    In the same way, EfeUOB has a character that can recognize ascorbic acid and taking Fe(II) in. EfeUOB is also controlled by Fur and when the density of Fe(II) in the cell is too high, Fur restricts EfeUOB and stops iron uptake.

    Using these characters, and by knocking down/out Fur and fieF, Fec/EfeUOB expression wouldn't be restricted so the amount of iron coming in would increase and the amount of iron going out would decrease. The system would work like when iron is lacking.

    As a result, the amount of iron inside E.coli would increase.

2.Materials&Methods

2.1.plasmid construct

プラスミドについて書く
    In order to magnetize E. coli, we thought to need to import as much Fe ions as possible in E. coli. So, we focused on ferritin that stored Fe irons. Ferritin is 24 mer composed by heavy chain and light chain. 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 H chain to L chain. Generally, the mammal ferritin has more L chain, on the other hand the ferritin derived from bacteria has more H chain. Because we wanted to store as much Fe as possible, the mammal ferritin is suitable for our theme. So, we decided to introduce human ferritin that can be expressed in E. coli.
    The ferritin has H chain and L chain but the ratio is different in the species. However, it may be depend on the expression. So, we constructed two different human ferritin; with middle RBS and with strong RBS. Further more, to regulate the expression and over expression, we used pBAD promoter. For these reasons, we constructed BioBrick based on BBa_I74608 (iGEM07 Cambridge).
Part link

2.2.Fur,fieFのノックダウン評価

Assay

3.Results & Discussion

3.1.plasmid建設の説明

3.2.Fur,fieFのノックダウン評価

4.Conclusion

それは・・