Http://2013.igem.org/Team:KU Leuven/Project/Glucosemodel/EBF

In this part, we will present you the insight of the E-β-farnesene (aka EBF) project. EBF is the most universal alarm pheromone that is released by almost all the 4000 aphid species in response to predation or other disturbances. The beneficial consequence among aphids due to EBF is presumed to be allowing the population taking evasive action after perception of EBF. Therefore the successful survival rate will be increased. In addition to the short term repelling effect, EBF can also cause the change in aphid’s development, fecundity, survival when introduced to different growth stages of aphid in a long term. Moreover, the natural predators of aphid such as ladybugs are attracted by EBF. During evolution, it seems that ladybugs have learned that the presence of EBF is linked to the presence of aphids.
Hence, we chose EBF as a major part in our design to perform the act of repelling the aphids. In the following sections, we will take you on a tour through the general background, the model and the genes, the wetlab work, and the biobricks we built for the EBF part.

We incorporated EBF synthase into the metabolic pathway of E. coli, with which, (2E,6E)-farnesyl diphosphate will be converted into (E)-beta-farnesene (EBF) and diphosphate as depicted in the reaction below.
The enzyme’s maxiamal activity will be achieved at a Mg²⁺ concentration of 5mM. The ideal pH for EBF synthase will fall in the range of 5.5-7. For the Artemisia annua protein the K_M is 0.0021mM, Kcat/K_M=4.5, turnover number 0.0095s^-1. For the Streptomyces coelicolor protein the K_M is 0.0168 and the turnover number 0.019s^-1.

The construct we designed for EBF project consists of constitutive promoter + lac operator + EBF synthase + double terminator (refer to project description). There are several organisms containing an EBF synthase gene, and we obtained two sources of this gene. One gene originates from Streptomyces coelicolor (Centre of Microbial and Plant Genetics of KUL) and the other one from Artemisia annua (sweet wormwood) (Brodelius P.; University Kalmar). However, the EBF synthase from Streptomyces coelicolor is a bifunctional enzyme (containing albaflavenone synthase activity), so the following build up of the final construct is based on EBF synthase gene from Artemisia annua.
For our construct, we chose a medium promoter with medium RBS (BBa_K608006), because we expect that high constitutive expression of EBF is not needed. For the double terminator, we decided to use BBa_B0015. The lac operator in front of the EBF synthase gene will play a role of switching the transcription of EBF synthase gene.

Gettin' the gene

To get the two sources of the target gene, we exploited different methods. In the case of EBF synthase gene from Streptomyces coelicolor, we got this gene with the help of colony PCR. In the story of EBF synthase gene from Artemisia annua, we received the gene in the pET28 vector from the research group in university of Kalmar. In this gene an additional EcoRI restriction site is present, which will conflict with the standard iGEM cloning work. Therefore we performed site specific mutation to get rid of this restriction site after transferring the gene into pSB1C3 backbone.

Cutting and pasting

After obtaining the target gene in the standard pSB1C3 backbone, we started our cloning work. The general concept we adapted is cutting the vector of promoter or terminator open, in which case the promoter or terminator is at the end of the linearized vector followed by ligate the insert into this vector. The reason why we undertake this strategy is because the size of promoter and terminator is so small, the possibility of false positives will be high if we use the 3A assembly method. In the situation of ligating the insert in front of the double terminator, we cut the vector of double terminator with EcoRI and XbaI restriction sites, cut the insert with EcoRI and SpeI restriction site. On the other hand, the promoter vector is cutted with SpeI and PstI restriction sites, and the insert is cutted with XbaI and PstI restriction sites.
The ligations were performed in two ways in parallel. In one setup we ligated for 20 minutes at 16℃, and in comparison, the second ligation of the same products is conducted at 16℃ overnight.
For transformation, we used both chemical competent cells and electrocompetent cells. Electroporation had higher efficiency when compared to heat shock transformation.

Confirmation

As soon as we observed colonies after transformation, we needed to confirm the products. The first step we did was usually a colony PCR to check if the insert is in the vector; however the colony PCR did not work every time. If it works, we will select the good ones to inoculate, otherwise randomly select some colonies to do the inoculation. The plasmid extraction was done on the overnight inoculated culture, and then we could confirm the size by digestion the plasmid. Only if the sample preceded these two confirmations, we will send the good ones to sequence, which will be the final confirmation.

G-blocks

Meanwhile, we also built the construct with lac operator in between the promoter and gene with gBlocks. We designed the gBlocks and assembled them together, followed by ligating the insert into pSB1C3 backbone. The positive colonies also needed to go through three confirmation steps mentioned above before we conclude we made them.

For more details of the labwork and the wetlab difficulties as well as how we overcame them, please consult the page of our wetlab journal.

Of course we met a lot of difficulties during the cloning work, but we kept trying different ways to overcome the obstacles, and finally we made the following bricks.

1. BBa_K1060001 This is a coding biobrick with the insert length of 1386bp, it is EBF synthase gene from Streptomyces coelicolor in pSB1C3 backbone.
2. BBa_K1060002 This is another coding biobrick with the insert length of 1725bp, it is EBF synthase gene from Artemisia annua in pSB1C3 backbone.
3. BBa_K1060008 This is an intermediate biobrick with EBF of Artemisia annua in front of double terminator.
4. BBa_K1060009 This is a generator biobrick with the insert length of 1924bp, it is medium constitutive expression of EBF synthase from Artemisia annua, in the pSB1C3 backbone.
5. BBa_K1060014 This is another generator biobrick with the insert length of 1923bp, it is strong constitutive expression of EBF synthase from Artemisia annua, in the pSB1C3 backbone.
6. BBa_K1060011 This is a generator biobrick with the insert length of 1965bp, it is medium constitutive expression of EBF synthase from Artemisia annua with lac operator after the promoter.