Part:BBa_K823023:Experience

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Applications of BBa_K823023

[http://2012.igem.org/Team:Groningen/international_cooperation Groningen 2012] verified that this backbone works!

[http://2016.igem.org/Team:Groningen/BrickCharacter Groningen 2016] showed how well it works, since we characterized the transformation efficiency:

Transformation Efficiency of B.subtilis plasmid backbone (BBa_K823023) created by iGEM LMU Munich 2012

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.

Results

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.

Fig. 1 Colonies after transformation of B. subtilis 168 trp+ with K823023. Concentration first row is 1 µg, second row is 100 ng, and third is 10 ng plasmid DNA.

Fig. 2 Colonies after transformation of B. subtilis 168 trp- with K823023. Concentration first row is 1 µg, second row is 100 ng, and third is 10 ng plasmid DNA.

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.

Fig. 3 Addition of lugol’s iodine to colonies grown on starch plates. There is only growth on the left plate 3,4 and 9. On the right plate there is only growth on 1,7 and 9. The other colonies might died from a too heat inoculation loop.

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.

Fig. 4 counting colonies after transformation.

For the counted colonies the transformation efficiency is calculated with the following formula:

(# Colonies on plate/ng of DNA plated) X 1000 ng/µg = CFU/µg of DNA

The amount ng of DNA plated, could be calculated with the following:

Volume of plasmid used in µL x concentration of DNA in ng/µl x (volume plated / total reaction volume)

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:

(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.

The results are summarised in the graphs below:

Fig. 5. Results of the transformation efficiency. The mean amount of colonies has been used to calculate the CFU/µg DNA.

Fig. 6 Results of the transformation efficiency. The mean amount of colonies has been used to calculate the CFU/µg DNA.

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.

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