Team:KU Leuven/Project/Glucosemodel/EBF

In this part, we will give some more information about the E-β-farnesene (aka EBF) part of the project. EBF is an alarm pheromone, released by almost all of the 4000 aphid species known thus far in response to the presence of predators (eg the ladybug) or other disturbances. In response to the produced EBF, aphids change their metabolism and turn into a winged form, allowing them to "flee the scene" and thus increase their survival rate. Apart from the short term repelling effect, EBF can also cause long term effects : changes in aphid’s development, fecundity, survival when introduced to different growth stages, etc. Moreover, natural aphid predators such as the ladybugs are attracted by EBF.
Hence, having our BanAphids produce EBF should help to repel aphids from our plant of choice. 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.

Since FPP is such an important precursor, used for the biosynthesis of lots and lots of compounds, it might turn out that once we insert a plasmid containing the β-farnesene synthase gene, we obtain only a very small amount of β-farnesene, since there isn’t enough FPP available for the required amount of β-farnesene we wish to produce.
A solution may be to implement plasmids which engineer a mevalonate pathway in E. coli, thereby upregulating the production of FPP. This larger amount of FPP may then be converted to β-farnesene, creating a large enough amount of this volatile for our uses. This was demonstrated many times by J.D. Keasling in S. cerervisiae, but Martin et al. (2003) implemented this mevalonate pathway in E. coli. In the article, they described their successful efforts to create a high level production of amorphadiene by introducing the mevalonate pathway in E. coli. They found that the expression of this heterologous pathway led to such an abundance of isoprenoid precursors that cells ceased to grow or mutated to overcome the toxicity. The authors state that because IPP and DMAPP are the universal precursors to all isoprenoids, the strains reported can serve as platform hosts for the production of any terpenoid compounds for which the biosynthetic genes are available.

It might be an option for us to ask J.D. Keasling for the plasmids pMevT and pMBIS and implement them into our E. coli along with the synthetic β-farnesene synthase gene, by which coexpression would result into high yields of β-farnesene.

We cloned and expressed the EBF synthase gene in E. coli, to break up (2E,6E)-farnesyl diphosphate into (E)-β-farnesene (EBF) and diphosphate (see reaction scheme below).
The enzyme prefers bivalent cations as cofactors; eg a Mg²⁺ concentration of 5 mM should be beneficial for EBF function. The ideal pH for EBF synthase will be between 5.5-7.

The EBF construct we designed consists of constitutive promoter with a lac operator, the EBF synthase itself and a double terminator. EBF is not only made by aphids but also by plants and other organisms in a form of bio-mimicry. We obtained two different sources of the EBF gene. One gene originates from the soil bacterium Streptomyces coelicolor (Centre of Microbial and Plant Genetics of KU Leuven). we chose this because it would be a perfect chassis for the ultimate expression of EBF in a bacterial setting. The other EBF gene is from the plant Artemisia annua (sweet wormwood) and was a kind gift from Professor Peter Brodelius (Kalmar University, Sweden). Here we were inspired with the plant origin. The K_M for the Artemisia annua protein 0.0021 mM, with a Kcat/K_M=4.5 and a turnover number of 0.0095 s^-1. For the Streptomyces coelicolor protein the K_M is 0.0168 mM and the turnover number 0.019 s^-1.
Unfortunately, the EBF synthase from Streptomyces coelicolor is a bifunctional enzyme, not only processing β-farnesene but also containing albaflavenone synthase activity. For this reason, we chose to follow up on the Artemisia annua gene and product. For our construct, our first choice was a medium strength promoter with medium RBS (BBa_K608006); we nonetheless also made the construct with a strong promoter and RBS. we used BBa_B0015 for the double terminator. The lac operator in front of the EBF synthase gene will play a role in switching on/off 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 linearised vector followed by ligating 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 cut with SpeI and PstI restriction sites, and the insert is cut 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 chemically competent cells and electrocompetent cells. Electroporation had a 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.

gBlocks

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.