Difference between revisions of "Part:BBa E0840:Experience"

Revision as of 22:10, 17 October 2014

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Please enter how you used this part and how it worked out.

Applications of BBa_E0840

BBa_E0840 is commonly used to quantify the function of transcriptional control devices.

igem2010 UT-Tokyo

We made an inverted version of this part, BBa_K313007, with the use of PCR.

GFP in the reverse direction are especially useful as outputs of genetic switches in which the coding regions of GFP are inverted by the recombinases.

User Reviews

UNIQ62e563192b26a4d6-partinfo-00000001-QINU

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Smelissali

Very visible even without UV excitation on plate and in solution after 24 hours incubation. Not as visible as Part:BBa_I13521 (mRFP), however.

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Reshma Shetty

BBa_E0840 was successfully used to quantify the behavior of BBa_R0040, BBa_J45992 and BBa_J45994.

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Aberdeen_Scotland 2009

Our miniprep, double and single digests worked as expected. We used this biobrick for building two of our parts (BBa_K182100 and 'BBa_K182101). We experienced very satisfactory cloning with high efficiency of transformants. The sequencing was correct. We discovered that all the bacteria containing this biobrick were releasing fluorescence even though there was no promoter upstream. The level of fluorescence observed was dependent on the plasmid copy number. We noticed that colonies displayed a visible, yellow-green colour, therefore selection was very easy.

UNIQ62e563192b26a4d6-partinfo-0000000B-QINU

Characterization

Transcriptional control of GFP generator

All three transcriptional control devices were quantified using BBa_E0840.

Growth phase dependent transcriptional control devices
We successfully designed, constructed and tested transcriptional control devices for constitutive, stationary phase dependent and exponential phase dependent protein production (A-C). To test and verify function of our three transcriptional control devices, we assembled each control device with the GFP protein generator BBa_E0840 and monitored the fluorescence of E. coli cultures with each device over time. For each device, we plot the change in fluorescence per unit time (normalized GFP synthesis rate) versus the cell density (OD600nm) (D). The constitutive transcriptional control device produced a high GFP synthesis rate irrespective of cell density. The stationary phase transcriptional control device produced a low initial GFP synthesis rate which increased with culture cell density. The exponential phase transcriptional control device produced an initially high GFP synthesis rate which dropped off as cell density increased. Data shown are averages of triplicate measurements of cultures grown from three individual colonies of each device. Error bars are the standard deviation of the three individual cultures.

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+The experiment to quantify GFP expression induced by Hg was made according to the protocol “Quantification of Green Fluorescent Protein (GFP) induced by different concentrations of mercury in Escherichia coli DH5α”.
+DH5α transformed with BBa_K1355002 was inoculated in LM (LB with low concentration of NaCl) liquid medium with chloramphenicol and grew until the Optical Density was 0.4 to 0.6abs (measured on spectrophotometer at 600 nm wavelength). After cell growth, an aliquot of 500μl in 5 eppendorf tubes (2ml) was taken and then added mercury chloride in order to achieve the concentrations of: 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml. The samples were incubated at 37°C on shaker. We collected each eppendorf tube at time 1 (01:30 hours of incubation), time 2 (03:00 hours of incubation) and time 3 (04:30 hours of incubation). Every sample was centrifuged at 12000g for 3 minutes and the pellet washed with TN Buffer (Nacl 0.15M + Tris HCl 10mM) and then re-suspended with 500μl of the same buffer. The same process was made to the bacterium without construction as a control to GFP expression/intensity. GFP expression was measured using the Hidex Chameleon spectrofluorimeter with excitation filter 340 nm and emission filter 500 nm wavelength. The Optical Density was measured simultaneously. All samples were analyzed in triplicate.
+The graph represented on Figure 1 shows the Optical Density of transformed DH5α with BBa_K1355002 in different Hg concentrations in function of time:
+[[File:bs1.png]]
+'''Figure 1.''' Optical Density measured in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+In these condition cell growth increases along time. The highest values correspond to bacteria not exposed to mercury or to small concentrations, as in 0 µg/ml, 0.01 µg/ml and 0.02 µg/ml. Suggesting a harmless condition to bacteria. However, cell growth decreases at higher concentrations as, 0.1 µg/ml, 0.2 µg/ml and especially 1µg/ml, giving to bacteria a hard time for development.
+The graph represented on Figure 2 shows the fluorescence emitted by DH5-alpha induced by different Hg concentrations in function of the time; and the graph represented on Figure 3, shows the ratio between fluorescence emitted and Optical Density.
+[[File:bs2.png]]
+'''Figure 2.''' GFP fluorescence intensity in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+[[File:bs3.png]]
+'''Figure 3.''' GFP fluorescence per cell growth ratio in the four given times, at mercury chloride concentrations of 0 µg/ml, 0.01 µg/ml, 0.02 µg/ml, 0.1 µg/ml, 0.2 µg/ml, and 1 µg/ml.
+Fluorescence can be observed in bacteria exposed to small concentrations, as 0.01µg/ml and 0.02µg/ml, but has low intensity. Fluorescence levels presents medium intensity in the higher concentration as consequence of cell death. Fluorescence intensity increased more than 480% in 0.2µg/ml concentrations, compared to fluorescence intensity from 1µg/ml, even in cell growth reduction, demonstrating its efficiency to induce mer promoter.
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