Team:Chiba/Project/uptake

Reprogramming Iron Homeostasis

1.Introduction

    To maximize the chance of magnetization, we would like to pump as much Fe into the cell as possible, and keep it. To this end, we tried to eliminate the negative controller (encoded by fur) on the Fec system (iron importer). Also, we tried to knock down the Fe exporter fieF.

Target-1:fur

    Fur (Ferric uptake regulator) controls multiple stages of iron metabolism such as taking iron in or storing iron. Major mode-of-action is in binding to Fur box to regulate transcription of various genes involved in the iron homeostasis. When cellular level of iron increase, Fur turns into the activated form, thereby start restricting the reproduction of the iron transporter (Fec system). Long story short, it ends up with down-regulating the iron uptake. At the same time, this active form Fur negatively controls the expression ofRyhB. RyhB is one of the sRNA that represses the expression of endogenous Ferritin. So, turning Fur in active form (by increasing cellular iron concentration) leads to the induction of ferritin construction.

When iron level get back, Fur turn back to inactive state, thereby restoring the iron uptaking machineries. RyhB would also work normally, so expression of ferritin is down-regulated to prevent iron depletion. On the other hand, other iron importing system EfeUOB recognizes rather ascorbic type and more specialized in importing Fe(II). EfeUOB is also controlled by Fur: When the level of Fe(II) becomes too high, Fur restricts EfeUOB and block uptaking the iron (II).

In summary, fur plays important roles iron homeostasis, and it is by nature a negative regulator for iron update. We would like to knockdown Fur so that iron transporter system would stay active even at the 'too much' situation. Possible downside could be that it also reduce the endogenous Ferritin formation, but we are constructing artificial (arabinose-inducible) system to functionally express human ferritins.

Target-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.

Part link

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.Function Check

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.

Fig. 2 CRISPRi efficiently silence transcription

Fig. 3 Function check about CRISPRi-lacZ

3.2.A knockdown of fieF or fur has no effect on iron uptake

Iron uptake was not detectable (iron concentration changes in media were less than micro molar order), even if fur or fieF was successfully knocked down.
A future subject is to experiment at more cell number of E. coli.

Fig. 4 Absorbance at each cell number of E. coli(BL21 and SHuffle® introduced each plusmid

Fig. 5 Absorbance of as a function of each iron concentration

Fig. 6