Difference between revisions of "Part:BBa K1927003"

Revision as of 21:02, 30 October 2016

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 –transpeptidase) 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.

Figure 1: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.

Functional validation of biobrick BBa_K1927003

The biobrick consist of an ampicillin resistant gene called ampR, and its sequence is collected from the pUC19 vector online. To functionally validating one of our brick we made the shipping vector pSB1C3 into an expression vector. We did a lot of research and decided to try the inducible promotor part J04500 https://parts.igem.org/Part:BBa_J04500 that came with the distribution kit. With this promotor upstream placed upstream of our gene of interest we could induce the transcription of our ampicillin resistant gene. Se drawing below:</p>

Figure 7: Illustration of the plan to combine the two parts
BBa_J04500 and BBa_K1927002 for expression of gene product
β-lactamase class A.

To generate our biobrick we used the 3A assembly protocol recommended by igem. https://parts.igem.org/Help:Protocols/3A_Assembly, where J04500 is part A and our ampR gene is part B. See protocol for more information.

To functional validate our brick we made overnight cultures of the hopeful colonies and plated them on agar plates containing IPTG (final volume of 1mM) and ampicillin. The plate was incubated overnight at 37 degrees. Colonies managed to grow on these plates, which confirm the biobricks beta lactamase activity.

The diagnostic tool
With an OD of ~1 we made a 16 and 64 times dilutions of the bacteria. This was to reduce the amount of bacteria so the sample is biologically relevant.

Graph 1: As observed for both the graph and the tables above the
absorbance at 486nm increases gradually with time.
The cleavage of Nitrocefin generates a red color in the sample,
which is visible by eye. The color will also depend on the amount of
bacteria in the sample as shown in graph 1. The 64 times diluted bacteria have a lower
absorbance in most of the time points.

Figure 8: Figure 1 displays the inside of our 3D model.
Cuvette to the left is synthetic urine and bacteria, but without Nitrocefin.
The cuvette to the right is same sample only with Nitrocefin.
This picture is taken after 20 minutes and it’s clear that the color change is visible by eye.

Sequence and Features