Part:BBa_K3152007
blaCMY10
We have removed the stop codon "TAA" from blaCMY10 sequence
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 143
Illegal BamHI site found at 259
Illegal BamHI site found at 364 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 226
Illegal NgoMIV site found at 341
Illegal NgoMIV site found at 1047
Illegal AgeI site found at 81 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 993
Usage and Biology
The BlaCMY-10 is part used in the β-Lactam degrading system project of iGEM team ASTWS-China 2019. The stop codon "TAA" of blaCMY10 was removed. This part is a β-lactamase，belonging to the group of ampC-related bla genes. The blaCMY-10 is constructed on pET24a. We chose pET24a because of its ideal structure, which contains an expression region of the coding strand transcribed by T7 RNA polymerase, and carries an optional C-terminal His Tag sequence. The protein expression is controlled by lac operator, which induced by IPTG. That means we can control the protein expression whenever we what, no antibiotic protein will be produced if there is no IPTG and the engineered E.coli with blaCMY-10 won’t be able to resistant β-lactams before induced. In order to find the most active and effective concentration of IPTG to induce the protein, a gradient test with the concentration of IPTG with 0.2mM, 0.4mM, 0.6mM, and 0.8mM was carried out. According to Figure 1A, it turned out that the optimum induced IPTG concentration was 0.2 mM. The blaCMY10 was purified by using BeyoGold™ His-tag Purification Kit. In Figure 1B, Lane 1 was loaded with protein ladder. Lane 2 and lane 3 contained the purified blaCMY10 protein (41.9 kDa), which compared with unpurified samples showed in lane 5 and lane 6. Showing that we purified blaCMY10 protein successfully.
Disc diffusion assay was applied to test the antibiotics performance of the blaCMY10 protein. β-lactamase was used as positive control, ddH2O was used as negative control, blaCMY10 was used as the test group. The columns were treated with three different kinds of β-lactam antibiotics(1 mg/mL ampicillin, 0.5 mg/mL cephalothin, 0.5 mg/mL cefoxitin). After 16 h incubation at 37℃, the test group showed no visible inhibition zone around the small paper discs, indicating that blaCMY10 effectively degraded the β-lactams (Figure 2).
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