Cloning

We performed all the cloning in E.coli, and did experiments on Asaia transformation, growth and resistance to antibiotics in parallel.

As the E. coli origin of replication does not work in Asaia, we added an origin that was compatible with this bacteria (which was recovered from a GFP-plasmid transformed into the Asaia we received from Italy). Conversely, Asaia's origin does work in E. coli, which is very useful as all cloning can be performed using standards developed for E. coli.

BioBricks

All our parts are in the pSB1C3 backbone vector (abbreviate C3).

First the legend:

• Ori = Origin of replication for Asaia and E. coli
• S = Strong Promoter
• W = Weak Promoter
• Immu = immunotoxin
• P25 = Pfs25 protein
• P28 = Pfs28 protein
• Kan = Resistance to Kanamycin
• Tet = Resistance to Tetracycline

This are the first and basic constructs we made. Just click on the part to get more information.

These are the BioBricks we obtained by combining the parts from above.

Here you get a global overview of our BioBricks construction plan.

Promoter characterization

To characterize the promoter we wanted to use in Asaia and E. coli we cloned it in front of a gene coding for CFP. By monitoring the CFP expression over time and comparing to a negative control that did not contain the CFP gene we could measure the activity of the promoter. We also recorded the growth of the two different cultures to see if expression would decrease the growth rate.
Both measurements were done at the same time in E. coli DH5a over 16 hours at 37°C and the results are averaged over 6 samples.
Unfortunately we can't compare our measurements with a well known promoter. Further characterization will focus on the measurement of the activity in Asaia.