Team:Chiba/Project/uptake

Uptake

1.Introduction

1.1.Fur

    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 RyhB.

1.2.fieF

    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

    We constructed four plasmids knocking down fur, fieF, gor, and trxB. at the same time, in order to confirm the function of dCas9, we constructed a plasmid knocking down lacZ.
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2.2.Evaluation of Fur,fieF knockdown

    We performed two experiments about CRISPRi system in order to confirm the knockdown function as desired. The purpose is a function check for lacZ, Fur, fieF, gor, and trxB.

Fig. 1 the method of function check about fur

Assay

2.3.Evaluation of absorbed iron volume

Experiment:

Fig. a

    E. coli stain BL21 was transformed by Plasmid shown in Fig. a . Then we cultured all transformants with atC.  atC was added to knock down fur and fieF.  After that we cultured it in the presence of ferric citrate, and measured the density of iron that weren’t taken in to E. coli and remained in the medium by measuring Absorbance (Abs 756) with color reagent.

3.Results & Discussion

3.1.plasmid建設の説明

3.2.Function Check

Fig. 2CRISPRi efficiently silence transcription

Fig. 3 Function check about CRISPRi-lacZ

1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated lacZ gene transcriptional silencing did not occur, resulting in blue-colored colony in which lacZ was expressed.
2) In the presence of aTc, CRISPRi-medeated lacZ gene transcriptional silencing successfully occurred, resulting in colorless colony in which lacZ was not expressed.
3) CRISPRi-medeated fur gene transcriptional silencing successfully occurred, resulting in the loss of kanamysin resistance.
    aTcがないときCRISPRi-lacZは発現されないためlacZの抑制は起こらず，X-galは分解されて培地は青く染まった。一方，aTcがあるとCRISPRi-lacZによる抑制が起こるため，lacZは発現されず，培地は白いままとなった。このことから，CRISPRi-lacZの機能を確認することができた。
    一方，fur, fieF, trxB, およびgor遺伝子を狙ったgRNAについて，その標的配列の下流にカナマイシン遺伝子を挿入した大腸菌株の作製を試みた。すると，furおよびtrxBの下流にカナマイシン遺伝子を挿入した株（それぞれMG1655Δfur::kmrおよびMG1655ΔtrxB::kmr）が得られた。一方，fieFおよびgorの下流にカナマイシン遺伝子を挿入した株（それぞれMG1655ΔfieF::kmrおよびMG1655Δgor::kmr）は得られなかった。これは，trxBおよびgorのプロモータが弱く，カナマイシン耐性遺伝子の発現量が，大腸菌にカナマイシン耐性を与えるほど多くないためと考えられる。

3.3.鉄取り込み量の評価

Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance

Fig. 4 BL21およびSHuffleでの、各プラスミドを導入した大腸菌の鉄試薬別のabsorbance
BL21、Shuffleともに、各細胞数において、鉄溶液の種類にかかわらず、吸光度にばらつきがでた。
これより、この実験方法では、大腸菌の取り込んだ鉄量が少なすぎるため、定量できなかったと考えられる。
今後の課題としては、さらに多い細胞数での定量、または、加える鉄試薬の濃度をさらに調整しなおす必要がある。
また、細胞破砕によって大腸内の鉄量を定量する方法も検討する必要がある。

4.Conclusion

それは・・