Part:BBa_K823023:Experience
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Applications of BBa_K823023
[http://2012.igem.org/Team:Groningen/international_cooperation Groningen] verified that this backbone works!
[http://2016.igem.org/Team:Groningen/BrickCharacter Groningen] showed how well it works, since we characterized the transformation efficiency:
The integration vector from team LMU Munich BacillusBiobrickbox
2012 (BBa_K823023) can be used to integrate an insert of interest
in B. subtilis. The cloned insert will be integrated within the
amyE locus in B. subtilis after transformation (see figure 1 for
the integration locus). The amyE gene encodes for the alpha-amylase
protein, which degrades starch. After transformation with
BBa_K823023, the AmyE locus will be interrupted by the insert. The
successful integration disrupts the ability of the bacteria to
degrade starch. Two strains of B. subtilis were chosen to test the
transformation efficiency - B. subtilis 168 trp+ and B. subtilis
168 trp-. Each strain was transformed with three different
concentrations of BBa_K823023; 1 µg/ml, 100 ng/ml, 10 ng/ml. This
experiment was done in triplicate. The transformation was
performed as indicated in Transformation of B. subtilis and colonies
were selected on LB agar plates containing 5 μg/ml chloramphenicol.
Subsequently 9 colonies were screened for correct integration of
K823023 with the starch test Integration check: Starch test. They were grown on
agar plates containing starch. The amylase activity of amyE is
visible as a clear zone (halo) after addition of lugol’s iodine
suggests the amyE gene was still intact and functional, which leads
to the conclusion that the integration was unsuccessful. The
colonies not forming the distinctive halo suggests a successful
integration into amyE, disrupting its amylase activity which
degrades starch. From the transformed bacteria suspension 50 µl were plated on LB
agar plates with 5 µl/ml chloramphenicol. All the plates had colony
formation, as seen in figure 1 and 2. After addition of lugol’s iodine, there were no clear zones
around any B. subtilis colonies, see figure 3. This result
demonstrated that the amyE locus in B. subtilis had been replaced
successfully. Apart from this transformation efficiency experiment,
our team has been using BBa_K823023 as a plasmid backbone for our
message and key for integration in B. subtilis. In addition, the colonies in the plates were counted. Plates
which contain a lot of colonies were divided in 16 areas as seen in
figure 4. The area with the estimated average amount of colonies
were counted. For the counted colonies the transformation efficiency is
calculated with the following formula: The amount ng of DNA plated, could be calculated with the following: The first calculation is given as example: 400 µl of bacteria suspension is transformed with 3,6 µl
plasmid. This plasmid had a concentration of 276 ng/µl. The plated
volume is 50 µl. The mean of the amount of colonies overnight was
1882,7 cfu. The result of the calculation is: The results are summarised in the graphs below: We can infer from the graphs that lower amount of DNA resulted
in higher colony forming units. The recommendation is not to use
1000 ng of DNA for transformation with BBa_K823023. Using 100 ng or
10 ng DNA for transformation would be slightly better. Transformation Efficiency of B.subtilis plasmid backbone
(BBa_K823023) created by iGEM LMU Munich 2012
Results
(# Colonies on plate/ng of DNA plated) X 1000 ng/µg =
CFU/µg of DNA
Volume of plasmid used in µL x concentration of DNA in
ng/µl x (volume plated / total reaction volume)
(3,6 µl * 276) * (50 µl / 403,6 µl) ≈ 123 ng DNA
plated
(1882,7 colonies / 123 ng plated DNA) * 1000 ng/µg =
1,5E+04 CFU/µg of DNA.
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