Part:BBa_K1979003
PBP5 coding device (promoter+PBP5+T7 terminator)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 135 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1178
Illegal BamHI site found at 168
Illegal XhoI site found at 1386 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 492
Illegal AgeI site found at 1094 - 1000COMPATIBLE WITH RFC[1000]
Construct:
The PBP5 sequence is cloned from the genome of E.coli through PCR, using the primers designed and synthesized based on the sequence of PBP5, with restriction enzyme sites EcoRI, XbaI, promoter BBa_J23101, rbs, and BamHI added on the upstream primer, and SpeI, PstI, T7 terminator added on the downstream primer before the SpeI site. The pSB1C3 backbone and PCR sequence are digested by EcoRI and PstI, after which they are ligated and become our new part.
Figure1. Double enzyme digestion check for BBa_K1979003. PBP5 has an expected size of 1200 bps (red box).
Proof of Function Through Experiments:
PBP5 causes the hydrolysis of tripeptide Lys-Ala-Ala(KAA) when active and not bound with penicillin, releasing one alanine and exposing one amino group.[1] We measure its hydrolysis by the amino group, which changes the color of KAISER, a substance that reacts with free amino acids to yield CO2, NH3, and an aldehyde, the NH3 further yielding a colored product (diketohydrindylidene-diketohydrindamine, a bi-indanedione derivative also called Ruhemann purple), yielding the color purple. With more penicillin present, more PBP5 becomes inactive, and the purple gets lighter.
Many poplypetides with a d-ala-d-ala end can be the substrate for PBP5, diacetyl-L-LysD-Ala-D-Ala (Ac2-L-Lys-D-Ala-D-Ala, Ac2-KAA) is the common substrate used to detect PBP5’s activity[2]. The PBP5 causes Ac2-KAA to hydrolyze, releasing one alanine and exposing one amino group. If bound with penicillin, PBP5 loses activity. And the protein is very sensitive to the penicillin in the liquid, so this mechanism is applicable as we try to determine the amount of penicillin in a low concentration system. the amino group, which turns Kaiser blue.
We mix 5 times diluted supernatant of the bacteria (treated with French pressure cell press and centrifugation at 12000rpm for 5min), with 100 times diluted Ac2-KAA. The mixture is equally devided into 8 centrifuge tubes, and different concentrations of penicillin (indicated below) are added. By applying the KAISER test, we were able to detect the amount of penicillin in each sample. (See protocol)
Results of KAISER Test
Interpretation:
According to the graph above, the horizontal axis represents the number of the sample and the vertical axis shows the optical density of each sample. By applying Pearson correlation coefficient, the result is 0.931032451, which means the two factors have strong correlation. As the concentration of penicillin increased, the value of optical density decreased, which proves the validity of our approach.
Reference:
1. R. F. Pratt Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins.(2008)
2. Qicun Shi, Samy O. Meroueh, Jed F. Fisher, and Shahriar Mobashery. Investigation of the Mechanism of the Cell Wall DD-Carboxypeptidase Reaction of Penicillin-Binding Protein 5 of Escherichia coli by Quantum Mechanics/Molecular Mechanics Calculations(2008)
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