Sharing and collaboration are core values of iGEM. With characterization of the production strain Vibrio natriegens we participated in an InterLab study and supported iGEM Team Marburg. Additionally, we recieved BioBricks from iGEM Team TU Darmstadt. Besides providing us the BioBricks, they gave us useful information about how to produce chitosan.
Vibrigens InterLab Study
The iGEM Team in Marburg is focussing on a new potential host for synthetic biology: Vibrio natriegens. V. natriegens can reach higher maximum specific growth rates than the current standard organism Escherichia coli. For the collaboration study our team performed the InterLab Study Vibrigens. This method ensures reproducibility of the experiments in different laboratories. The procedure of this experiment is comparable with the iGEM InterLab Study.
The aim of the experiment was measuring fluorescence and OD600 of V. natriegens cells transformed with different devices.
Results and Discussion
First, electrocompetent V. natriegens cells were prepared, aliquoted and stored at -80 °C. The competent cells were used for transformation of six different devices, additionally one positive and one negative control (table 1).
Table 1: Devices and corresponding Part Number used in the experiment.
First colonies on the plates appeared 7 to 8 hours after plating. For further measurements two transformed colonies of each device were picked and inoculated for overnight culture. The next day, OD600 was measured. The cultures were diluted to a target OD600 of 0.02 and incubated for another 3 hours. Samples were taken at 0 and 3 hours. OD600 (figure 1) and fluorescence (figure 2) were measured afterwards. All measurements were performed with BioTek Synergy 2 plate reader with a 96-well black flat bottom plate with clear bottom.
Figure 1: OD600 of V. natriegens after 0 and 3 h incubation in LB + v2 + 2 µg/mL Chloramphenicol.
Figure 2: Fluorescence (Excitation 485 nm, Emission 528 nm) of V. natriegens after 0 and 3 h incubation in LB + v2 + 2 µg/mL chloramphenicol.
In figure 2 the increase of the fluorescence after 3 h incubation is shown in all devices. The devices 1, 4 and 5 are clearly more fluorescent, so the expression of the fluorescent protein is higher. This might be caused by a stronger promotor activity in these devices compared to the positive control and the other devices.
The results of the Vibrigens InterLab Study are comparable with the results of the iGEM InterLab.
iGEM Team TU Darmstadt
Very much in line with the iGEM philosophy we utilized an existing BioBrick (developed by TU Darmstadt 2017 ChiTUcare) as starting point for our novel approach. Thus we utilized the chitin synthase (NodC, BBa_K2380002) and two different chitin deacetylases (NodB, BBa_K2380041 and COD, BBa_2380043). We were happy to build on their experience and vision, applying chitosan as an antimicrobial coating, similar to their implementation of it in their hydrogel for the use of wound healing patches. In agreement with last years team members from Darmstadt, we have received the relevant BioBricks as plasmid DNA from team TU Darmstadt 2018 (combimer).
In several chats via e-mail and personal communication at the European Meetup in Munich they provided us useful information concerning the expression and purification especially of the deacetylase NodB. This particular enzyme poses some difficulties on the purification process as it is produced in inclusion bodies. Therefore, we concentrated on COD as chitin deacetylase.
Thanks to iGEM Darmstadt we saved a lot of time on the construction of the initial basic parts for our chitosan subteam. Furthermore, we are glad to share our parts and experience with team Darmstadt for further development of a chitosan producing genetically engineered machine.