Part:BBa_K2317002

CbeA

CbeA-CbtA is one of the Escherichia coli TA systems.CbtA inhibits the polymerization of bacterial cytoskeletal proteins, MreB and FtsZ. The fission of bacteria will be suppressed but the growth of bacteria is normal. Cbea was found to directly interact with MreB and FtsZ, and enhance the bundling of their filamentous polymers in vitro. Besides, CbeA can relieve the toxicity of CbtA. (Fugure 1.)

Figure 1. The mechanism of CbeA and CbtA(type IV TA system).

Usage and Biology

The population and growth condition can be reflected by Abs600, so through the curve of Abs600-time, the effect of CbeA and CbtA towards bacteria can be displayed. Abs600 values were measured from 0 to 14 hours in two groups and the toxication curves were drawn as shown in Figure 2. The growth rate of vector group bacteria after adding L-Arabinose sped up for a short time and then slowed down. At last, the trend was consistent with that without L-Arabinose. However, TA group, with the addition of L-Arabinose, showed the same OD and Abs600 values from the 4th hour for a while. After that, Abs600 values displayed a little increase.

Figure 2. (A) Different concentrations of L-Arabinose were added into vector group and the growth curve was drawn to made sure the effect of L-Arabinose towards bacteria. According to the results, the addition of L-Arabinose would first slightly enhance and then suppress the growth. However, this effect would disappear after a while. (B) Different concentrations of L-Arabinose were added into TA group and the growth curve was drawn to made sure the effect of CbtA(induced by L-Arabinose) towards bacteria. According to the results, the addition of L-Arabinose the growth would be stagnated at first and then recover.

Besides, after culturing for 14 hrs, difference in turbidities of bacteria in different groups was visible Figure 3.

Figure 3. (A) and (B) The two figures were taken to compare the turbidities of TA group after 14 hours od culturing, and the difference could be told by eyes.

Characterization:

Through agarose gel electrophoresis, the plasmid we constructed was verified.(figure 4.) We chose DL 10000 as the marker and used BBa_K864402 as the control. Eight of the lanes contained pSB1C3-CbtA monoclones and Eight of the lanes contained pSB1C3-CbeA monoclones. After that, we sent them for sequencing and the results showed that the sequences were right.

Figure 4. The result of agarose gel electrophoresis.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 116
  
• 1000
  COMPATIBLE WITH RFC[1000]