Difference between revisions of "Part:BBa K4195039"

Latest revision as of 09:37, 13 October 2022

ampC

Biology

AmpC
β-lactamase (AmpC) is a bacterial enzyme that facilitates resistance against β-lactam antibiotics by hydrolyzing the β-lactam ring, deactivating the antibiotic. It can also catalyze the hydrolysis reaction of nitrocefin, resulting in a distinct color change from yellow to red(1).
Fig. 1 The color transformation catalyzed by AmpC.

Usage and Design

β-lactamase was selected for its good performance in enhancing the report signal. We add BBa_K3222000, BBa_K4195068, BBa_B0034 and BBa_K731721 to construct the expression system and obtained the composite BBa_K4195153, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Characterization

1. In Vivo Verification

1) Agarose Gel Electrophoresis
After transferring the plasmid into BL21(DE3), colony PCR was used to certify the plasmid was correct. The expected result was obtained.
Fig. 2 The result of colony PCR. Plasmid pSB1C3.
2) Absorbance measurement
Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of AmpC is observed by measuring the absorbance in 482 nm as time progressed using microplate reader.
Results are documented in related pages: BBa_K4195156, BBa_K4195157, BBa_K4195158, BBa_K4195160, BBa_K4195161, BBa_K4195164, BBa_K4195165, BBa_K4195166.

2. In Vitro Verification

Plasmid was put into the cell-free system for expression. The expression behavior of AmpC is observed by measuring the absorbance in 482 nm as time progressed using microplate reader.
Results are documented in related pages: BBa_K4195159, BBa_K4195162, BBa_K4195163.

3.Identification and protein Purification

In order to get AmpC and further purify it, we added a His-tag (6×His) to the C-terminal of AmpC. The coding sequence of AmpC-his was cloned into the expression vector pET-28a(+) by Gibson assembly. Then we transformed the plasmid into E. coli BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Fig. 3 DNA gel electrophoresis of the colony PCR products of AmpC-his_pET-28a(+).
After being cultivated and induced by IPTG, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of AmpC-his (Fig. 3), the target protein (32.4 kDa) can be observed at the position around 35 kDa on the purified protein lanes (FR).

Fig. 4 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples. Target bands (32.4 kDa) can be observed at the position around 35 kDa.

4. Paper-based colorimetric assay

In order to determine the corresponding relationship between protein concentration and absorbance, we used nitrocefin as the substrate to perform a colorimetric assay. The filter paper was cut into circular pieces, and the substrate was spotted onto them. After that, a short video was taken while we add purified AmpC to the pieces. With the protein concentration from high to low, a color change from orange red to brilliant yellow can be observed (Fig. 4).

Fig. 5 The result of paper-based colorimetric assay. 0.5 mg/mL nitrocefin was added (3 μL per piece).

Reference

1. K. E. Boehle, C. S. Carrell, J. Caraway, C. S. Henry, Paper-Based Enzyme Competition Assay for Detecting Falsified β-Lactam Antibiotics. ACS Sens 3, 1299-1307 (2018).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 284
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 7: / Line 7: @@
 ''β''-lactamase (AmpC) is a bacterial enzyme that facilitates resistance against ''β''-lactam antibiotics by hydrolyzing the ''β''-lactam ring, deactivating the antibiotic. It can also catalyze the hydrolysis reaction of nitrocefin, resulting in a distinct color change from yellow to red(''1'').
 [[File:T--XMU-China--Ampc.png|500px]]<br/>
-'''Fig. 1 The color transformation catalyzed by AmpC'''
+'''Fig. 1 The color transformation catalyzed by AmpC.'''
 ===Usage and Design===
@@ Line 19: / Line 19: @@
 After transferring the plasmid into BL21(DE3), colony PCR was used to certify the plasmid was correct. The expected result was obtained.
 [[File:T--XMU-China-K4195153_(K4195153_pSB1C3,_colony_PCR).png|300px]]<br/>
-'''Fig. 1 The result of colony PCR. Plasmid pSB1C3'''<br/>
+'''Fig. 2 The result of colony PCR. Plasmid pSB1C3.'''<br/>
 '''2) Absorbance measurement''' <br/>
 Colonies harboring the correct plasmid were cultivated and induced. The expression behavior of AmpC is observed by measuring the absorbance in 482 nm as time progressed using microplate reader.  <br/>
@@ Line 30: / Line 30: @@
 In order to get AmpC and further purify it, we added a His-tag (6×His) to the C-terminal of AmpC. The coding sequence of AmpC-his was cloned into the expression vector pET-28a(+) by Gibson assembly. Then we transformed the plasmid into ''E. coli'' BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.<br/>
 [[File:T--XMU-China-K4195153_his11.png|300px]]<br/>
-'''Fig. 2 DNA gel electrophoresis of the colony PCR products of AmpC-his_pET-28a(+).'''<br/>
+'''Fig. 3 DNA gel electrophoresis of the colony PCR products of AmpC-his_pET-28a(+).'''<br/>
 After being cultivated and induced by IPTG, GE AKTA Prime Plus FPLC System was employed to get purified protein from the lysate supernatant. Purified protein was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining. As shown in the gel image of AmpC-his (Fig. 3), the target protein (32.4 kDa) can be observed at the position around 35 kDa on the purified protein lanes (FR).<br/>
 [[File:T--XMU-China-K4195153_sds.png|300px]]<br/>
-'''Fig. 3 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples.''' Target bands (32.4 kDa) can be observed at the position around 35 kDa.<br/>
+'''Fig. 4 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the elution samples.''' Target bands (32.4 kDa) can be observed at the position around 35 kDa.<br/>
 ====4. Paper-based colorimetric assay====
 In order to determine the corresponding relationship between protein concentration and absorbance, we used nitrocefin as the substrate to perform a colorimetric assay. The filter paper was cut into circular pieces, and the substrate was spotted onto them. After that, a short video was taken while we add purified AmpC to the pieces. With the protein concentration from high to low, a color change from orange red to brilliant yellow can be observed (Fig. 4).<br/>
 [[File:T--XMU-China-K4195153_his.png|300px]]<br/>
-'''Fig. 4 The result of paper-based colorimetric assay.''' 0.5 mg/mL nitrocefin was added (3 μL per piece).
+'''Fig. 5 The result of paper-based colorimetric assay.''' 0.5 mg/mL nitrocefin was added (3 μL per piece).
 ===Reference===
 . K. E. Boehle, C. S. Carrell, J. Caraway, C. S. Henry, Paper-Based Enzyme Competition Assay for Detecting Falsified β-Lactam Antibiotics. ''ACS Sens'' '''3''', 1299-1307 (2018).