Difference between revisions of "Part:BBa K1927003:Experience"
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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> | 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> | ||
| − | + | https://static.igem.org/mediawiki/2016/7/78/T--UiOslo_Norway--Igeneschetch.jpg | |
<p class="boxnotes leftext imgcap"><i> | <p class="boxnotes leftext imgcap"><i> | ||
Figure 7: Illustration of the plan to combine the two parts <br> | Figure 7: Illustration of the plan to combine the two parts <br> | ||
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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. </p> | 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. </p> | ||
| − | + | https://static.igem.org/mediawiki/2016/d/d2/T--UiOslo_Norway--table1.jpg | |
| − | + | https://static.igem.org/mediawiki/2016/d/da/T--UiOslo_Norway--classAGRAPH.jpg | |
<p class="boxnotes leftext imgcap"><i> | <p class="boxnotes leftext imgcap"><i> | ||
Graph 1: As observed for both the graph and the tables above the <br> | Graph 1: As observed for both the graph and the tables above the <br> | ||
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absorbance in most of the time points. </i></p> | absorbance in most of the time points. </i></p> | ||
| − | + | https://static.igem.org/mediawiki/2016/e/e1/T--UiOslo_Norway--3ddevice.jpg | |
<p class="boxnotes leftext imgcap"><i> | <p class="boxnotes leftext imgcap"><i> | ||
Figure 8: Figure 1 displays the inside of our 3D model. <br> | Figure 8: Figure 1 displays the inside of our 3D model. <br> | ||
Latest revision as of 12:50, 20 October 2016
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how you used this part and how it worked out.
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:
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.
User Reviews
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