Part:BBa_K1468004:Experience

Applications of BBa_K1468004

For testing the behaviour of BBa_K1468004, 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 wild type ones were plated on LB with the appropriate antibiotic (chloramphenicol) and LB respectively.

- Overnight incubation at 37°C.

- Bacterial colonies were transfered to 1 mL of LB and LB supplemented with chloramphenicol (depending on which we are refering to, wild type or transformants).

- 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 or LB (both supplemented with doxycyline for pTet promoter induction). Four biological replicates were perfomed.

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

Two aliquots were made:

- one of them was used to measure the OD at 595 nm. - The other one was sonicated, centrifuged (cell dibries elimination) and incubated with XGal for 6 minutes (the reaction was stopped by adding Na2CO3). A blue colour measurement was carried out (OD at 630 nm).

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

The results obtanied were:

Figure 1.Behavior of Bba_K1468004 in six different E. coli strains. Absorbance values were corrected by the absorbance displayed by non-transformed cells. As it can be observed the expression is similar in all the E. coli strains.

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