Part:BBa_K1927003
Beta - lactamase AmpR
This part is an ampicillin resistant gene with the J04500 inducible promotor.
Usage and Biology
The mechanism of beta lactamases are oriented to the bacterial cell wall. This cell wall is unique to bacteria and consist of several components. Gram Positive and gram negative bacteria will have a different cell wall composition. In general, Gram-positive bacteria have a thicker layer of cell wall as well as a layer of cytoplasmic membrane. These layers consist of several conserved compounds such as monomeric disaccharide tetrapeptide, which are usually also those that will trigger an immunological defence respons of the host. Gram-negative bacteria (e.g., Escherichia coli) typically contain an outer membrane, an intervening periplasmic space where a thin layer of cell wall resides, and a layer of cytoplasmic membrane. Beta lactamases are usually produced both by gram negative and positive bacteria, either from plasmid or chromosomally. Beta lactamases are able to resist several types of antibiotics. These antibiotics all have in common a 4 - atom ring called beta lactam ring which the enzyme are able to hydrolyze and break open and the molecule looses its antibacterial function. Penicillin, a regulary used antibiotic have such a beta lactam ring. This drug was the first antibiotic to be discovered and is still widely used today. This ring will bind to an enzyme (DD âtranspeptidase) that is in charge of renewing the bacterial cell wall. Without this enzyme there will be no new formations of peptidoglycans for the cell wall and the integrity of the bacterial cell wall will be lost, it will eventually rupture and the bacteria will die. By hydrolyzing the ring, it will make the molecule unable to bind to the cell wall producing enzyme, thus the Penicillin have lost its destructive activity. Read more about usage and biology for beta lactamases on BBa_K1927002.
Applications of BBa_K1927003
The UiO team have submitted a similar part <a href="https://parts.igem.org/Part:BBa_K1927002">BBa_K1927002</a> But since the pSB1C3 vector is not an expression vector, we could not use this for calibrating our diagnostic tool. We therefore looked into the iGEM library and found BBa_J04500 which has a ribosomal binding site and IPTG inducible promotor. By combining AmpR gene downstream for the BBa_J04500 we could theoretically express the gene product which is a β-lactamase class A. This was done by restriction digestion of part BBa_J04500 with Spel and Pstl and the AmpR-insert with Xbal and PstI. Restriction digest was followed by ligation and transformation of TOP10 cells and grown on LB agar plate with chloramphenicol. Colony PCR was performed to verify successful ligation with our insert. Restriction digestion of miniprepped DNA was performed to confirm prior to sequencing.
Figure3: Gel analysis of colony PCR with BBa_K1927003. The insert was inserted into the shipping vector pSB1C3 by using 2 different approaches; 3A assembly and a single restriction digest followed by ligation. Lane 1 and 10 are DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific). Lane 2 and 11 are positive control (validated part BBa_K1927000). Lane 3-7 are different colonies from 3A assembly. Lane 8-9 and 12-14 are restriction digest and ligation approach. Lane 4, 6-8, 12-13 shows the appropriate band at approx. 1000bp.
Figure 4: Restriction digest of miniprepped DNA with restriction enzymes Pstl and Xbal. Lane 1 shows DNA ladder (GeneRuler 1 kb DNA ladder, Thermo Fisher Scientific), while lane 1-7 shows the same digest with miniprepped DNA derived from different bacteria colonies (transformed competent TOP10 cells with BBa_K1927003). Lane 1, 6 and 7 shows the appropriate bands with the insert at approx. 1000bp and vector at approx. 2000bp.<i/>
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 942
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