Part:BBa_K2913024

ftnA-M

In order to enhance the tumor-targeting ability of our engineered bacteria, we select a mutant ferritin, ftnA-M to create a magnet responding cassette, so that we can reduce the whole bacteria mass in our project, minimizing the potential harmful effects of bacteria on the human body.

Actually, the inherent ferritins of E. coli creat an inner cavity storing iron in a hydrated amorphous form of iron oxide which is biocompatible and magnetic depending on its crystal structure. However, the mineralized iron stored inside natural ferritins exhibits poor crystallinity which facilitates iron release in times of need but also results in a very modest inherent magnetic moment.

According to the article we consulted, the engineered ferritins including H34L + T64I, named ftnA-M, is doubly mutated at the “B-channel” (red circle in Fig. 1) that transports iron, likely enlarging the B-type channel, exhibited the strongest ferromagnetism. [1]

Fig. 1. 3D structure of ftnA-M protein[1]

We expected to verify the increased magnetotactic ability of engineered E. coli, the experiments we carried were described as followings: We constructed the gene circuit shown as Fig. 2.

Fig. 2. Gene circuit of magnet targeting cassette.

Usage and Biology

Targeting and self-regulation module Magnet Targeting cassette:

=Method:=

=Method:=

1. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analysis of protein expression levels of engineered E. coli BW25113:

(1) Transformation of the plasmids into E. coli BW25113

(2) Expression of ftnA-M was induced in cells by adding 0.2% arabinose to LB liquid medium during log-phase growth (OD600nm reached 0.4), simultaneously exposed the cells to 100 μM iron (II) sulfate as supplement.

(3) Cultured the induced bacteria for 6 hours at 16 ℃ in a shaker.

(4) Quantified the intensity of OD600nm reaching 6, used ultrasonic waves to break the cell, 100 ℃, boiled for 5min. Centrifuged, and discarded the precipitate.

Result of SDS-PAGE is shown in Fig. 4.

2. We used RbFeB magnet to verify the increased magnetotactic ability of engineered E. coli BW25113:

(1) Inducing expression of ftnA-M protein method to magnetize the cells was described above (1(1)-(3)).

(2) Centrifuged, resuspended the cells with M9 liquid medium with the total M9 medium volume of the experimental and control group was respectively up to 20 ml in 50ml centrifugal tube.

(3) Added 0.2% arabinose, 100 μM iron (II) sulfate as supplement. Set the position of RbFeB magnet between the 50ml centrifugal tube of experimental group and control group. (Fig. 3.) Cultured the engineered E. coli and E. coli for 24 hours.

Fig. 3. Image of the experiment settings. The RbFeB magnet was set between the 50ml centrifugal tube of experimental group (right) and control group (left).

Result of verification of magnetism is shown in Fig. 5.

Result

[1]. The result of SDS-PAE indicated that the ftnA-M protein was expressed as we expected.

Fig. 4. SDS-PAGE analysis to evaluate protein expression of ftnA-M. Lane 1: E. coli BW25113, Lane 2: ftna-M/E. coli BW25113.

2. The engineered E. coli showed the aggregation in the magnetic side at the slope bottom of the tube while the control group of E. coli BW25113 did not show obviously aggregation.

Fig. 5. Image of cells cultured in M9 liquid medium for 24 hours. 0.2% arabinose, 100 μM iron (II) sulfate as supplement were added and The RbFeB magnet was set between the 50ml centrifugal tube of experimental group (right) and control group (left).

Reference

[1]Liu, X., et al., Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evoluton. Sci Rep, 2016. 6: p. 38019.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]