Difference between revisions of "Part:BBa K300002:Experience"

Revision as of 19:57, 27 October 2010

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Applications of BBa_K300002

This part was tested through BBa_K300086.

Methods

Inoculum (into 5 ml LB+Amp) from glycerol stock of:

BBa_K300086
BBa_K300088
BBa_K300090
BBa_K300099
BBa_K173000 (positive control)
BBa_B0031 (negative control)

Cultures were grown ON at 37°C, 220 rpm.

The following day cultures were diluted 1:100 and let grow again for about five hours at 37°C, 220 rpm.

The optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.

Then we performed a 21-hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted.

Results

Raw growth curve

Raw GFP curve

Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one -BBa_E0040)

Culture	Doubling time [min.] ± std error
BBa_K173000	76.3336 ± 1.4362
BBa_K300086	73.6685 ± 1.6245
BBa_K300088	74.8806 ± 2.7699
BBa_K300090	75.9433 ± 3.6808
BBa_K300099	78.4634 ± 2.5622
BBa_B0031	70.8421 ± 2.2181

Discussion

All cell cultures showed a similar growth curve; doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to cells.

From GFP curve it's possible to appreciate that in BBa_K300086, BBa_K300088, BBa_K300090, BBa_K300099 GFP accumulation it's very similar and it's significantly different from that of negative control BBa_B0031. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded.

The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.

User Reviews

UNIQ03924bbce6bd372e-partinfo-00000013-QINU

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UNIPV-Pavia iGEM 2010

This part was used as a head domain alone and to construct the following synthetic composite affinity tags:

UNIQ03924bbce6bd372e-partinfo-0000001A-QINU

@@ Line 61: / Line 61: @@
 ====Discussion====
-All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to cells.
+All cell cultures showed a similar growth curve; doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to cells.
 From GFP curve it's possible to appreciate that in <partinfo>BBa_K300086</partinfo>, <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded.