Difference between revisions of "Part:BBa I13521:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
how you used this part and how it worked out. | how you used this part and how it worked out. | ||
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| + | ===Applications of BBa_I13521=== | ||
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| + | 2011iGEM SJTU-BioX team has modified RFP to obtain a new reporter. | ||
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===Applications of BBa_I13521=== | ===Applications of BBa_I13521=== | ||
Revision as of 03:20, 6 October 2011
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_I13521
2011iGEM SJTU-BioX team has modified RFP to obtain a new reporter.
Applications of BBa_I13521
For parts BBa_I13521 and BBa_I13522, their excitation and emission maxima were determined. BBa_B0034 was used as a negative control. These data were used later in following characterization experiments as set parameters. The host was E. coli Top Ten in all experiments.
The graph above shows the emission scan for RFP in part BBa_I13521. Since it was the best option in terms of fluorescence yield, 570 nm was set as the excitation wavelength. As it can be seen from the graph, maximum emission wavelength is 605 nm.
Meanwhile, the graph below shows the emission scan for GFP in part BBa_I13522. Since it was the best option in terms of fluorescence yield, 395 nm was set as the excitation wavelength. As it can be seen from the graph, maximum emission wavelength is 515 nm.
For the parts BBa_I13521 and BBa_I13522 which are mentioned above, confocal laser scanning microscope images were taken in the host Top Ten.
Characterization was performed by METU-TURKEY IGEM2010 Team
Thermostability Assay
For this BioBrick, the denaturation temperature was determined by heating the protein at a range of temperatures, and then measuring the fluorescence. This is a useful characterisation as it allows the selection of an appropriate reporter gene for the required temperature.
Stock solutions of mRFP were prepared by extracting the protein from cell lysate, and then 50μl aliquots of the solution were heated in a PCR thermocycler along a temperature gradient.
After two hours, 30μl was removed from each aliquot and diluted with 170μl of 20mM Tris buffer to give 200μl samples. The fluorescence of the samples was then measured on a 96-well plate. The corresponding curve was plotted on the graph below.
Results of the heat denaturation experiment. The temperature at which half of the protein is denatured is measured by looking at its fluorescence (PTm50) mRFP1: 82.2°C; GFPmut3b: 61.6°C; Dendra2: 89.1°C; sfGFP: 75.0°C.
The sigmoidal curves that were calculated gave us the following function which also created the coefficient K which happens to relate to PTm50 (temperature at which half of the protein is denatured measured by looking at its fluorescence):
This shows that the protein denatures at around 81°C
.This characterisation was performed by the Imperial College London 2011 Team.
User Reviews
UNIQ07e24dbbab2552ef-partinfo-00000003-QINU
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melissali |
Very visible to the naked eye (vs. CFP, YFP were not very visible without UV excitation). |
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UNIPV-Pavia iGEM 2011 |
NB: All tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C in low copy plasmid pSB4C5. This strain constitutively expresses TetR, so pTet promoter can be induced with aTc.
PTet promoter was assembled upstream of different mRFP coding sequences, containing an RBS from the Community collection; these are the measurement devices used:
The assembled RBSs are here reported with their declared efficiency:
For an inducible device, the RBS variation has the purpose to stretch the induction curve, thus modulating its PoPs-OUT range. The complex RBS-promoter acts as a whole regulatory element and determines the amount of translated protein. RBSs have been reported to have an un-modular behavior, since the translational efficiency is not independent on the coding sequences, but variates as an effect of different mRNA structure stability [Salis et al., Nat Biotec, 2009]. It is not possible to separate the effects of the sole promoter and of the sole RBS on the total amount/activity of gene product (in this case study, mRFP). For this reason, every combination 'Promoter+RBS' was studied as a different regulatory element. Regulatory elements were characterized using mRFP reporter protein for different RBSs in terms of Synthesis rate per Cell (Scell) and R.P.U.s (Relative Promoter Units) as explained in measurements section.
The Hill function relating the induction to the Scell is reported below: Scell=α * ( δ + (1-δ)/(1+(K/Induction)η) ) Operative parameters of the promoter are derived from the estimated Hill equations obtained by nonlinear least squares fitting (lsqnonlin Matlab routine) of the Hill function expressed in RPUs:
The protocols for the characterization of pTet promoter are reported in the pTet measurement section. The data collected from the mRFP measurement systems were processed as described in data analysis section. The induction curves were obtained by fitting a Hill function as described in modelling section and the estimated parameters for pTet are reported in the pictures and in table below. This promoter is widely studied and characterized usually using the strong RBS BBa_B0034. Here we have characterized its transcriptional strength as a function of aTc induction (ng/ml) for different RBSs. Three different induction curves were obtained and are reported in figure: 
The estimated parameters of the Hill curves described in the figures are summarized in the table below:
The measurement system pTet-B0031-mRFP-TT couldn't be assayed because its fluorescence output is under the detectability threshold of our measurement instrument. For this reason, the parameters of the corresponding Hill curve couldn't be estimated and are reported as 'Not Determined' ND. α parameter (representing the maximum trascriptional rate in the studied range of induction) varies as expected with the RBS variation and also the δ and η parameters are quite different among the RBS variations. The kpTet parameter is quite constant among the RBS variations, thus suggesting that in this case the RBS variation doesn't substantially affect the switch point of the Hill curve, even if the amplitude and the maximum slope are not quite maintained (for the η parameter, maybe fitting problems). The operative parameters are summarized in the table below:
From these parameters, it is evident that whilst the switch-point is almost maintained for all the RBSs, the linear boundaries are similar for RBS30 and RBS32 but for RBS34 are moved on the right of one order of magnitude. |
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