Introduction

We look throughout the registry and we did not find any part containing a Nanoluc coupled with a T7 promoter. Although there is another well characterized Nanoluc parts with a pBAD promoter, there is none with T7. That is why, our team wanted to complete and improve the registry by creating a new one.
Moreover, we noticed that the BBa_K1159001 (a nanoLuc construction from Munich-2013) characterization missed the concentration of arabinose to add for the induction, in order to get the same results, which are really good. This was quite problematical if we wanted to obtain the same relative light unit. In our case, a consequent luminescence is essential in order to visualize our bacteria.

Light intensity has been measured 5 hours after induction with arabinose or IPTG depending on the Biobrick, when DO 600 was between 0,6 and 0,8. Bba_K325909 already has the substrate synthesis within its sequence, therefore we only had to measure the kinetic during 5h of induction and took the maximum value. For the others, we added NanoGlow substrate from Promega (ratio 1:1), measured kinetics over 120 min and took the maximum value.

Construction of NanoLuc plasmid

Improvement of BBa_K1159001 Biobrick Characterization

In the same experiment, we also improved the characterization of BBa_K1159001. We observed that no references were performed for the characterization of the BBa_K1159001. That is why, we wanted to compare the first Luxbrick created by Cambridge in 2010, to this first NanoLuc under pBad promoter. BBa_K1159001 showed a light intensity approximately 140 times brighter than BBa_K325909 at equivalent inducer concentration.

Relative Light Unit depending on percentage of arabinose

As mentioned above, we also further characterized the NanoLuc construct of iGEM Munich 2013 by analyzing its expression under different percentage of arabinose. BBa_K1159001 light intensity is linked to the percentage of arabinose. The light intensity increases from 0.01% to 1% of arabinose. However, over 1% of arabinose, the intensity does not increase anymore.

Holin Endolysin

Our kill switch was designed to work in a system like pDawn, a light-inducible expression system. However, this system works with reversed sequence. That is why we designed a simple reversed Biobrick Holin-Endolysin (HE). This part was inserted into native pDawn for characterization of the Biobrick.

Bacteria expressing pDawn-HE were prepared in a pre-culture until DO600 reached a value between 0,6 and 0,8. Then, one condition was illuminated with white-light and incubated at 37°C, another one was kept in the dark and incubated at 37°C and the last condition was illuminated with white-light and incubated at room temperature. The value of DO was taken regularly to observe the kinetic of growth under each condition.

In absence of light, and at 37°C, bacteria growth was normal but light illumination was enough to slow-down the growth of bacteria. Incubation at room temperature with white-light illumination allowed better folding of HE and had a stronger effect on the growth of bacteria.

Bubble formation and detection

Our system of microfluidic chip is designed in a way that it has 2 entries and 1 exit. The entries allow the fluids to enter the system and they enable both physical and informatical engines to control the movement of the fluids (water and blue colorant in one entry and oil with a really low viscosity in an other one).
After a suitable positionning of the chip under the microscope according to the “Fluigent” design, the player can begin the game by creating a blue bubble of the mixture water and colorant in the main fluid: the oil. In our prototype of the Bio-Console, this bubble does not contain any enginneered bacteria, but as we firstly imagined it, it should have contained one.

However, we wanted to insert a red filter in order not to activate the biological systems (VVD construction and liposomes) as well as a white flash that could be triggered by the informatical code of the game when it is game over. Eventually we didn’t have enough time to set this up. Indeed, the creation and implementation of the code took longer than expected as a consequence of some difficulties to manage to realize a code able to detect the bubble at the beginning of the game. Moreover, the association of the code and the Fluigent’s technology also took longer than expected.

Nevertheless, we achieved our goal to create a prototype of the Bio-Console and our system works as expected. In addition, the absence of the systems we imagined in order not to activate the biological systems can be overcomed by the use of bioluminescence as the way of vizualisation.