Difference between revisions of "Part:BBa I723020:Experience"

Revision as of 15:55, 1 October 2011

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Applications of BBa_I723020

User Reviews

UNIQa922b11c2ca67698-partinfo-00000000-QINU

BBa_I723020 1 Not understood Sebastian Murmann ETH Zürich 2011

We had some troubles to get the part out of the iGEM spring distribution 2011. Although trying for several times, we did not succeed in transforming the plasmid provided in the distribution. Thus we used the plasmid pCK04YxylR as source for the promoter. We further modified the part by adding a double stop codon to it, resulting in BBa_K625002. The second modification was to remove the remaining sequence of xylU, which resulted in the short promoter variant BBa_K625003. The characterization and comparison of the new promoters can be found in the characterization section of BBa_K625002 respectively BBa_K625003.

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 <I>Sebastian Murmann ETH Zürich 2011</I>
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+We had some troubles to get the part out of the iGEM spring distribution 2011. Although trying for several times, we did not succeed in transforming the plasmid provided in the distribution. Thus we used the plasmid ''pCK04YxylR'' as source for the promoter. We further modified the part by adding a double stop codon to it, resulting in [https://parts.igem.org/Part:BBa_K625002 BBa_K625002]. The second modification was to remove the remaining sequence of ''xylU'', which resulted in the short promoter variant [https://parts.igem.org/Part:BBa_K625002 BBa_K625003]. The characterization and comparison of the new promoters can be found in the characterization section of [https://parts.igem.org/Part:BBa_K625002 BBa_K625002] respectively [https://parts.igem.org/Part:BBa_K625003 BBa_K625003].
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-We had some troubles to get the part out of the iGEM spring distribution 2011. Although trying for several times, we did not succeed in cloning the plasmid provided in the distribution. Thus we used the plasmid ''pCK04YxylR'' as source for the promoter. We further modified the part by adding a double stop codon to it, resulting in [https://parts.igem.org/Part:BBa_K625002 BBa_K625002]. The second modification was to remove the remaining sequence of ''xylU'', which resulted in the short promoter variant [https://parts.igem.org/Part:BBa_K625002 BBa_K625003]. The characterization and comparison of the new plasmids can be found in the characterization section below.
-'''Characterization'''
-'''Experimental setup'''
-[[Image:DSC_0146.JPG|100px|left|thumb|'''Figure 1: Experimental setup''' of XylR experiment.]]
-''E. coli'' strain JM101 was transformed with two plasmids containing the transcriptional regulator XylR, the degradation cassette ''xylMABN'' and a GFP reporter coupled to [https://parts.igem.org/Part:BBa_K625002 BBa_K625002] respectively [https://parts.igem.org/Part:BBa_K625003 BBa_K625003]. For the first two of those we used the plasmid pCK04AxylR according to [[#Ref1|[1]]]. The reporter plasmid we constructed ourselves by using [https://parts.igem.org/Part:BBa_K625005 BBa_K625005] as a backbone.
-We inoculated 50 mL of LB medium with an overnight culture of the co-transformed strains and set the OD<sub>600</sub> to 0.1. Upon reaching the exponential growth phase, the cultures were induced with ''m''-xylene. Therefore we put a sterile test tube with ''m''-xylene into the flask and sealed it with parafilm in order to get an air-induced response.
-The samples were taken 3 hours after induction. OD<sub>600</sub> and GFP fluorescence was measured then in a 96-well plate. Afterwards the data was normalized in order to get more consistent results.
-'''results'''
-[[Image:Pu promoter test final colour.png|200px|right|thumb|'''Figure 2:  Characterization of BBa_K625002 and BBa_K625003''' ''m''-xylene was added by air-induction.]]
-We could observe a clear increase in GFP fluorescence when ''m''-xylene was added in a test tube. Thus GFP production can be induced by ''m''-xylene present from the air. The results of the experiments can be seen in ''fig. 2''. By comparing [https://parts.igem.org/Part:BBa_K625002 BBa_K625002] and [https://parts.igem.org/Part:BBa_K625003 BBa_K625003], we can see that the leaky expression of GFP is higher in the shortened promoter [https://parts.igem.org/Part:BBa_K625003 BBa_K625003] compared to the longer one. There are several possible reasons for this observation. Most likely it is due to the drop of the natural part downstream of the promoter, which is usually also included in Pu.
-Nevertheless, we could also see a higher level of induction in the setup with [https://parts.igem.org/Part:BBa_K625003 BBa_K625003] (around 7-fold) compared to the [https://parts.igem.org/Part:BBa_K625002 BBa_K625002] setup (around 5-fold). Thus both of the provided promoters can be used for transcriptional regulation with XylR.
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