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 contribute to the detoxification of cell from various chemicals. It is known that the knock out of this fieF result in increase in iron accumulation ⁴ . Interestingly, this efflux pump is reported to be out of the complex regulation network of iron homeostasis (not regulated by fur at all).


Thus, by temporary knocking down Fur, the expression of 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.

2. Experimental Results

2.1. Toward Blick for temporal/ inducible KO of the target gene

Because of the physiological impact to the host cell, we decided not to go for normal, permanent removal of the target genes (fur/ fieF) in the genome. Also, the gene knock-out (via genome editing) is chassis engineering, not the BioBrick construction. To best enjoy the portability of plasmid (BioBrick), it is ideal to make plasmid-coded gene-knockout system. To this end, we decided to establish temporal knockdown systems using recently developed CRISPRi system.
We constructed four plasmids for the knocking down each of fur, fieF, gor, and trxB. Alongside, we also constructed a plasmid knocking down lacZ.
The key regulating molecule (the mutant dCas9) is expressed in trans using another plasmid. Expression of dCas9 is induced by the addition of anhydro Tetracycline (aTc).

Fig. 1 the method of function check about dCas9

Assay

2.2. CRISPRi as inducible knockdown platform

We tested CRISPRi system for the aTc-inducing knockdown for the model targeted site lacZ. 1) In the absence of anhydro Tetracycline (aTc), CRISPRi-medeated lacZ gene transcriptional silencing is not supposed to occur. Indeed, we observed blue-colored colonies in aTc (-) plate. This means lacZ is well expressed. 2) In the presence of aTc, CRISPRi-medeated lacZ gene transcriptional silencing should occur. Yes, we saw drastic decrease in color of colony in aTc (+) plate. Thus, the expression of lacZ is repressed as we designed.

Fig. 2 Function check about CRISPRi-lacZ

2.3. Inducible knockdown of targeted locus

Then we tried to knockdown our target site Fur. 1) To confirm the effective knock-down of the gene, we engineered a strain that has kanamycin resistance gene right after the target sequence (within the reading frame of fur).: See Fig1.
2) CRISPRi-medeated fur gene transcriptional silencing was performed by expressing dCas9 together with engineered snRNA targeted to fur.
3) The resultant transformant was resistance to kanamysin (see left panel of Fig. 3), but it shows kanamycin sensitivity upon addition of aTc. This clearly shows that the CRISPRi system is effectively silencing the transcription of the targeted locus (fur).

2.4. The effect of knockdown of fur on iron uptake

Experiment: E. coli stain BL21 transformed with Plasmid shown in Fig. 4 was cultured in the presence of aTc. Then the transformant was cultured in the media containing ferric citrate. The conc. of iron left in the media was measured by titration using specialized indicator for irons.

Results: In summary, we did not observe any increase in iron uptake, even in the condition that fur or fieF was successfully silenced. We want to combine all the knock-down guiders for fur and fieF in hope for the synergistic effect on ion uptake/ storage. Also, we would like to combine these system with ferritin-expressing devices we developed alongside. Also, putting all systems into Biobrick is also our hope to finish soon (without any problem: in next couple of weeks, we are done! -note on Sept 27th).

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. 3CRISPRi efficiently silence transcription

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