Part:BBa_K4195108

I0500-B0034-mepS-B0015

The enzyme can lower the stability of Gram-negative bacterial membrane structure. We use BBa_I0500 to construct the hypervesiculation composite part BBa_K4195108.

Biology

MepS is a PG endopeptidase, which is related to OMV massive secretion. The PG-OM cross-linking of Gram-negative bacteria can be altered via localized modulation of PG degradation and synthesis with its participation(1).

Usage

The promoter (BBa_I0500), RBS (BBa_B0034), mepS gene (BBa_K4195004) and terminator (BBa_B0015) were assembled into pSB1C3 plasmid backbone to get the composite part BBa_K4195108 (Fig. 1) and express the enzyme MepS. Then the ligation mixture was transformed into E. coli DH5α & E. coli BL21(DE3), and the positive transformants were confirmed by chloramphenicol, colony PCR and sequencing.

Fig. 1 Gene circuit of mepS.

Characterization

1. Agarose gel electrophoresis (AGE)

When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-2254bp (lane K4195108).

Fig. 2 The result of colony PCR. Plasmid pSB1C3.

2. Hypervesiculation ability

Then, the colony with the corrected sequence was cultivated to verify its hypervesiculation ability. We purified and quantitated the OMV of experimental and control group, whose gene circuit only contains the promoter BBa_I0500. The experimental result is shown on Fig. 3. Observing the difference of OD₆₀₀ between two groups, we can guess that bacterial membrane structure’s stability decreases in experimental group and draw the conclusion that the composite part BBa_K4195108 has the hypervesiculation ability compared with control group.

Fig. 3 Normalized protein concentration and OD₆₀₀ of experimental and control group.

3. Enzyme kinetics

Also, in order to explore the enzyme reaction kinetics of MepS, different time nodes were set after the induction of L-arabinose. The cell supernatant was separated immediately by centrifuging after measuring OD₆₀₀ in 0, 0.5, 1.0, 2.0, 4.0, 6.0 h to stop the growth process. Then we purified and quantitated OMV of different time nodes and drew the image of normalized protein concentration and time after induction (Fig. 4). The conclusion can be easily drawn that the degree of enzyme reaction increases to saturation with time.

Fig. 4 Enzyme kinetics image of MepS.

Reference

1. C. Schwechheimer, D. L. Rodriguez, M. J. Kuehn, NlpI-mediated modulation of outer membrane vesicle production through peptidoglycan dynamics in Escherichia coli. Microbiologyopen. 4, 375-389 (2015).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961