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Assembly Plan

Background

Communication using quorum sensing

Quorum sensing (QS) is the communication tool of bacteria. It is usually done through secretion and detection of small signal molecules or proteins. Depending on the bacterial density, these molecules can trigger a change of behaviour, i.e. invoke a function only when there are enough bacteria present in that one area. Quorum sensing can control several things, from biofilm formation to bioluminescence. The most commonly known and most explored QS system is the Lux system, which was originally isolated from Vibrio fischeri, where it controls the production of luciferase, an enzyme that aids in the production of a yellow light.

Using the pathogens own quorum sensing system to detect and track its source

When we decided that we wanted to fight pathogens it became obvious that we needed a way to detect and track them. After doing some research, we decided that this could be done by hijacking the pathogen’s own quorum sensing. Naturally, the first thought that came to our minds was to use the Lux system from V. fischeri. However, this had been done before in 2008 by Heidelberg and it also did not provide the specificity that we wanted for our system. We decided to dig deeper and see if we could find a more unique quorum sensing system. Among some potential candidates was Yersinia enterocolitica, which uses a quorum sensing system called the Yen system.

The Yen system

As it turns out Y. enterocolitica has a homologous quorum sensing system to the famous Lux system, the Yen system. From this system we chose to steal two parts. A recognition region, the yenbox, fused together with a promoter and an activator, YenR, that can recognise and interact with the yenbox. When YenR binds to the yenbox it induces the expression level of the promoter fused to the yenbox. Later, in the presence of Y. enterocolitica, its signaling molecules, 3-oxo-hexanoyl homoserine lactone (OHHL), will start flowing into our system, interacting with YenR. When binding occurs between OHHL and YenR, YenR will lose its active shape and thereby its ability to interact with the yenbox. The induction will then be lost and the expression level will return to its base level. ^[1]

By BioBricking and characterizing this system, we wish to make it possible to get a Y. enterocolitica density triggered response. Our goal with this system is to control our two other systems, the Targeting and the Killing system.

System Design

While stealing the yenbox together with the wild type promoter fused with it, we had no idea about the strength of the wild type promoter or if it would even work at all. Because of this, we created an alternative version where we replaced the wild type promoter with a standardised one (J23113). In the wild type version there is an overlap between the wild type promoter and the yenbox. Hence, we mimicked the same while creating our customized version where we had an overlap between the promoter and the yenbox. Since the strength of the promoter would correspond to the leakage in our system, we wanted to have a weak promoter to minimize the leakage. We chose the constitutive promoter J23113 (

BBa_J23113

) from the Anderson library. Unfortunately, the promoter J23113 did not begin with the same two bases as the end of the yenbox. We were left with the option of either changing the two bases in the yenbox sequence or changing the two bases in the sequence of the promoter J23113. In the article by Ching-Sung Tsai and Stephen C. Winanas ^[1] they discovered that the binding between the activator YenR and the recognition region of the yenbox is not dependent on the entire sequence of the yenbox. Depending on which part of the yenbox that is changed or replaced, YenR binds to the yenbox with different strengths. However, it still interacts with the yenbox and induces the strength of the promoter. Based on this fact, together with the knowledge that the Anderson promoters are very sequence dependent, we chose to change two bases in the sequence of the yenbox.

We also stole the coding sequence ^[2] of the activator YenR, from Y. enterocolitica, which we codon optimized for E. coli using a web tool ^[3] and synthesized it together with the RBS B0034 (

BBa_B0034

). Since we always want production of the activator YenR, it was coupled to three different constitutive promoters from the Anderson promoter library with three different strengths.

Result

For characterisation we created the constructs

BBa_K1381008

and

BBa_K1381009

, where the yenbox fused with a promoter was coupled to the green fluorescent protein (GFP). These constructs were then cloned into the backbones pSB1C3 and pSB3C17 and transformed into competent E. coli cells already containing one of the YenR constructs BBa_K1381005, BBa_K1381006 or BBa_K1381007 on the backbone pSB1K3. The double transformed cells were then streaked on plates containing both the antibiotic Kanamycin and Chloramphenicol and left it overnight to grow. Cells containing only the constructs yenbox_promoter-B0032-GFP were also streaked and left to grow.

The following day, overnight cultures were prepared and left for 16 h to grow into stationary phase. After that, 10 µL of the overnight culture was put into 500 µL of PBS solution and left for one hour for stabilization. The green fluorescence production was then measured using a flow cytometer. The results of the test is shown below. Graph 1 above, shows the induction relative to the base level of expression. In graph 1.A. we can observe the induction but cannot predict to what extent. This is because these cells are transformed with two high copy plasmids containing the same ori(origin of replication). The consequence will be that the cells will confuse the two plasmids with each other and have no control of in what amount the two respective plasmids are present. It will only ensure that it is 100-300 plasmids present in total, regardless of which plasmid it is. This is why we also cloned the yenbox containing constructs into a low copy plasmid with a different ori than the high copy ones.

In graph 1.B., it can be seen that the amount of YenR that is produced is correlated, as expected, to the rate of the induction. When coupled to the strongest of the three promoters, J23102, the production is increased up to five folds.

In graph 1.C., we did not observe any induction This implies that our customized version of the yenbox fused with the promoter J23113 did not work as intended. It seems like the activator YenR could not recognise nor interact with the yenbox due to the changes made in the last two bases of the yenbox. It should be tested to redesign this part so that the yenbox is not fused to the promoter but just simply put beside it without any overlap. If the induction is not extremely distance dependent, that might work better. Otherwise, you could try to find other promoters, were the first two bases match with the two last bases of the yenbox, so that we don’t have to modify the yenbox.

These kind of constructs were also constructed with a blue fluorescence protein (BFP) that were also transformed into cells already containing one of the YenR constructs BBa_K1381005, BBa_K1381006 or BBa_K1381007 on the backbone pSB1K3. They were then plated and restreaked together with a non-fluorescence containing reference and a base level reference without any YenR. These plates were used to see if the induction could be seen in UV light, with the naked eye. Unfortunately, as you can see in fig. 1 below, this was not the case. The color could be seen, but there was barely any difference in the intensity of the different cells.



It should also be mentioned that we did an attempt to create bigger constructs containing the characterisation constructs with the yenbox coupled to GFP and the YenR constructs (yenbox_promoter-B0034-GFP-terminator-promoter-B0034-YenR). But for some reason these assemblies did not work. After some troubleshooting we came to the conclusion that it was too stressful for our bacteria to produce both BFP and YenR on the same high copy plasmid or that these constructs were not stable in E. coli and got rejected or mutated by the cell.

• [1] Ching-Sung Tsai and Stephen C. Winanas, The quorum-hindered transcription factor YenR of Yersinia enterocolitica inhibits pheromone production and promotes motility via a small non-coding RNA, 2011, Molecular Microbiology 80[2], 556-571
• [2]http://www.ncbi.nlm.nih.gov/nuccore/123440403/?from=1798138&to=1798872
• [3] http://eu.idtdna.com/CodonOpt