Coding

Part:BBa_K1927003:Experience

Designed by: Marthe Jrgensen   Group: iGEM16_UiOslo_Norway   (2016-10-14)
Revision as of 12:27, 20 October 2016 by Marthejj (Talk | contribs) (→‎Applications of BBa_K1927003)


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Applications of BBa_K1927003

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:

<img class="goldphoto" src="T--UiOslo_Norway--Igeneschetch.jpg"/>

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.

<img class="respic" src="T--UiOslo_Norway--table1.jpg"/> <img class="respic" src="T--UiOslo_Norway--classAGRAPH.jpg"/>

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.

<img class="goldphoto" src="T--UiOslo_Norway--3ddevice.jpg"/>

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.

User Reviews

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