Difference between revisions of "Part:BBa K625002"
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<partinfo>BBa_K625002 short</partinfo> | <partinfo>BBa_K625002 short</partinfo> | ||
| − | This is an adapted version of the Pu promoter [https://parts.igem.org/wiki/index.php?title=Part:BBa_I723020 BBa_I723020]. The original design contains the RBS and first 81bp of the XylU. To prevent unwanted fusion proteins from emerging (when this BioBrick is cloned without scar), a double stop codon was inserted at the end of the promoter. | + | '''This is an adapted version of the Pu promoter [https://parts.igem.org/wiki/index.php?title=Part:BBa_I723020 BBa_I723020]'''. The original design contains the RBS and first 81bp of the XylU. To prevent unwanted fusion proteins from emerging (when this BioBrick is cloned without scar), a double stop codon was inserted at the end of the promoter. |
=='''Characterization'''== | =='''Characterization'''== | ||
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==Experimental setup== | ==Experimental setup== | ||
| − | [[Image:DSC_0146.JPG| | + | [[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. | ''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. | ||
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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. | 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. | + | 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== | ==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. | |
Latest revision as of 15:58, 1 October 2011
Pu promoter long version with stop codon
This is an adapted version of the Pu promoter BBa_I723020. The original design contains the RBS and first 81bp of the XylU. To prevent unwanted fusion proteins from emerging (when this BioBrick is cloned without scar), a double stop codon was inserted at the end of the promoter.
Characterization
Experimental setup
E. coli strain JM101 was transformed with two plasmids containing the transcriptional regulator XylR, the degradation cassette xylMABN and a GFP reporter coupled to BBa_K625002 respectively BBa_K625003. For the first two of those we used the plasmid pCK04AxylR according to [1]. The reporter plasmid we constructed ourselves by using BBa_K625005 as a backbone.
We inoculated 50 mL of LB medium with an overnight culture of the co-transformed strains and set the OD600 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. OD600 and GFP fluorescence was measured then in a 96-well plate. Afterwards the data was normalized in order to get more consistent results.
results
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 BBa_K625002 and BBa_K625003, we can see that the leaky expression of GFP is higher in the shortened promoter 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 BBa_K625003 (around 7-fold) compared to the BBa_K625002 setup (around 5-fold). Thus both of the provided promoters can be used for transcriptional regulation with XylR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 195
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
[1] [http://aem.asm.org/cgi/content/abstract/64/2/748 S. Panke, J. M. Sanchez Romero, V. de Lorenzo: Engineering of Quasi-Natural Pseudomonas putida Strains for Toluene Metabolism through an ortho-Cleavage Degradation Pathway , Applied and Environmental Microbiology, 1998, Vol. 64, No. 2]