Difference between revisions of "Part:BBa K4195004"
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The promoter (<partinfo>BBa_I0500</partinfo>), RBS (<partinfo>BBa_B0034</partinfo>), mepS gene (<partinfo>BBa_K4195004</partinfo>) and terminator (<partinfo>BBa_B0015</partinfo>) were assembled into pSB1C3 plasmid backbone to get the composite part <partinfo>BBa_K4195108</partinfo> (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.
 
The promoter (<partinfo>BBa_I0500</partinfo>), RBS (<partinfo>BBa_B0034</partinfo>), mepS gene (<partinfo>BBa_K4195004</partinfo>) and terminator (<partinfo>BBa_B0015</partinfo>) were assembled into pSB1C3 plasmid backbone to get the composite part <partinfo>BBa_K4195108</partinfo> (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.
  
−
[[File:T--XMU-China--fig.1 I0500-mepS circuit.png|300px]]
+
[[File:T--XMU-China--fig.1 I0500-mepS circuit.png|300px|center]]
  
 
'''Fig. 1 Gene circuit of ''mepS'''''.
 
'''Fig. 1 Gene circuit of ''mepS'''''.
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When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-2254bp (lane K4195108).
 
When we were building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-2254bp (lane K4195108).
  
−
[[File:T--XMU-China--fig.2 BBa K4195108 colony PCR BL21(DE3).png|300px]]
+
[[File:T--XMU-China--fig.2 BBa K4195108 colony PCR BL21(DE3).png|300px|center]]
  
 
'''Fig. 2 The result of colony PCR. Plasmid pSB1C3.'''
 
'''Fig. 2 The result of colony PCR. Plasmid pSB1C3.'''
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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 <partinfo>BBa_I0500</partinfo>. The experimental result is shown on Fig. 3. Observing the difference of OD<sub>600</sub> between two groups, we can guess that bacterial membrane structure’s stability decreases in experimental group and draw the conclusion that the composite part <partinfo>BBa_K4195108</partinfo> has the hypervesiculation ability compared with control group.
 
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 <partinfo>BBa_I0500</partinfo>. The experimental result is shown on Fig. 3. Observing the difference of OD<sub>600</sub> between two groups, we can guess that bacterial membrane structure’s stability decreases in experimental group and draw the conclusion that the composite part <partinfo>BBa_K4195108</partinfo> has the hypervesiculation ability compared with control group.
  
−
[[File:T--XMU-China--fig. 3NC vs mepS-OMV secretion.png|300px]]
+
[[File:T--XMU-China--fig. 3NC vs mepS-OMV secretion.png|300px|center]]
  
 
'''Fig. 3 Normalized protein concentration and OD<sub>600</sub> of experimental and control group.'''
 
'''Fig. 3 Normalized protein concentration and OD<sub>600</sub> of experimental and control group.'''
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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 OD<sub>600</sub> 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.
 
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 OD<sub>600</sub> 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.
  
−
[[File:T--XMU-China--fig.4 Dynamics-Meps.png|300px]]
+
[[File:T--XMU-China--fig.4 Dynamics-Meps.png|300px|center]]
  
 
'''Fig. 4 Enzyme kinetics image of MepS.'''
 
'''Fig. 4 Enzyme kinetics image of MepS.'''

Revision as of 02:55, 11 October 2022


mepS


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.

T--XMU-China--fig.1 I0500-mepS circuit.png

Fig. 1 Gene circuit of mepS.


Characterization

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

T--XMU-China--fig.2 BBa K4195108 colony PCR BL21(DE3).png

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


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.

T--XMU-China--fig. 3NC vs mepS-OMV secretion.png

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


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.

T--XMU-China--fig.4 Dynamics-Meps.png

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