Difference between revisions of "Part:BBa K1468000:Experience"

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'''Figure 1.''' Behavior of Bba_K1468000 in six different ''E. coli'' strains. Fluorescence intensity of Bba_K1468000 was corrected by the fluorescence of cells containing an empty plasmid.
 
'''Figure 1.''' Behavior of Bba_K1468000 in six different ''E. coli'' strains. Fluorescence intensity of Bba_K1468000 was corrected by the fluorescence of cells containing an empty plasmid.
  
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== BBa_K1468000 stability ==
 
  
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37°C and 200 rpm are the standard growth conditions of ''E.coli''. Varying these conditions (Temperature, pH, salinity, ultraviolet radiation exposure and vacuum) and measuring the growth after three hours, (O.D.595) as well as BBa_K1468000 (fluorescence), was used to test the effect on BBa_K1468000 expression (all data are the average of at least three measurements; fluorescence values are normalized to OD595).
 
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The results obtained were:
 
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https://static.igem.org/mediawiki/2014/6/65/Bb1pLDM2.png
 
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'''Figure 2. ''' The strain XL1 Blue transformed with a Biobrick which encodes GFP (BBa_K1468000) was grown at different temperatures in a thermocycler for three hours, the cells were collected and O.D600 was measured. Fluorescence was also determined and compared to the non-transformed/wildtype or control strain. As the plot shows, Biobrick expression is affected by growth at different temperatures, fluorescence (normalized to O.D600) is higher around ''E. coli'' optimal growth temperature (37-41 °C); over or below this rather short temperature range fluorescence decreases.
 
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https://static.igem.org/mediawiki/2014/5/5d/Bb1pLDM3.png
 
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'''Figure 3. ''' BBa_K1468000 expression under different temperatures, including temperature fluctuation (from 37°C to 41°C, every minute each) in a thermocycler. Controls were incubated at 37 and 41°C.  After an incubation period of 3 hours, O.D.600 alongside fluorescence was measured. The results show that the expression of the Biobrick is not affected by cycling temperatures.
 
  
  

Latest revision as of 15:30, 13 October 2014

Applications of BBa_K1468000

For testing the behaviour of BBa_K1468000, we used six different E. coli strains: DH5α, XL1Blue, JM109, DH10B, HB101, BL21(DE3); which were grown in standard conditions: 37°C, 200 rpm (revolutions per minute) and LB with a standard composition (1% NaCl, 1% peptone and 0.5 % yeast extract).

To carry out the assay, we did as follow:

- Transformed strains -with Bba_K1468000 and with the empty plasmid, no biobrick- were plated on LB with the appropriate antibiotic (chloramphenicol).

- Overnight incubation at 37°C.

- Bacterial colonies were transfered to 1 mL of LB supplemented with chloramphenicol.

- A 20 minutes at 37°C incubation was carried out.

- These precultures (100 µL) were used to inoculate a tube containing 2 mL of LBC. Four biological replicates were perfomed.

- The cultures were incubated (standard conditions) until OD595 is between 0.2-0.4 (1 hour aprox.).

- The OD at 595 nm was measured.

- GFP fluorescence (λex= 493nm; λem=505nm) was measured.

With all this data the average, fluorescence /cell density, and standard deviation were calculated taking into account the four biological replicates.

The results obtanied were:

Figure 1. Behavior of Bba_K1468000 in six different E. coli strains. Fluorescence intensity of Bba_K1468000 was corrected by the fluorescence of cells containing an empty plasmid.



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