Our main objective
Our main goal is to achieve the production of synthetic bacteria with two plasmids of interest, for a major production of resveratrol from by-products of the wine industry waste.
On one side, we will introduce a plasmid with three genes involved in the resveratrol synthetic pathway (TAL, 4CL and STS), so as to empower the bacteria with the ability to synthesise resveratrol from precursors p-cumaric acid and malonil-CoA. On the other side, our E.coli will also contain another plasmid, responsible for resveratrol precursor malonil-CoA overexpression. The minor generation of malonil-CoA in the cell and its consumption in the fatty acids synthetic pathway justifies the construction of this second plasmid, which aims to overexpress the ScACC enzyme that converts acetil-CoA to malonil-CoA.
1. Plasmid with TAL, 4CL and STS
The resulting synthetic bacteria will be able to increase resveratrol production from p-cumaric acid and malonil-CoA, both precursors obtained from by-products of the wine industry waste.
The obtaining of the final plasmid with TAL, 4CL and STS results from the previous construction of three vectors containing each one gene. Once the three individual plasmids are obtained, the extraction of each gene is needed in order to unify them in a final vector. The final plasmid will then be introduced into the bacteria.
The cloning method used for all these constructions is the Golden Gate Method (1):
The following procedures are required to obtain this plasmid with TAL, 4CL, STS, ppor.
All protocols are available in the "protocols" section of the wiki:
Competent cells preparation and heat shock transformation
Obtaining competent cells from an E. coli starter culture is a key step to allow the bacteria to absorb DNA, which will recombine with their genome. Afterwards, the competent cells are transformed by heat shock introduction of ampicillin resistance PJUM plasmid.
Miniprep, electrophoresis and enzymatic digestion of PJUM plasmid
The extraction from the competent cells of the DNA plasmid corresponding to pUC19 vector is carried out following the miniprep protocol. The extracted fragments are analysed by gel electrophoresis, to ensure the correct extraction of the vector.
Once the pUC19 plasmid is obtained, it is digested with the BsaI restriction enzyme. Thereafter, a gel electrophoresis is required to verify the digestion process.
Efficiency measurement
Efficiency measurement allows us to check the effectiveness of bacterial transformation. Its calculation takes into account the number of colony-forming units as well as the amount of transformed DNA to be expressed in the corresponding units.
\[Efficiency = {\frac{FCU}{ug DNA}}\]
We can be sure that we have good efficiency if the values are higher than \[10^{9}\frac{FCU}{ug DNA}\]
Synthesis of promoter, RBS, ppor, terminator.
Due to erroneous bacterial transformation on Ampicillin-resistant plates of the parts corresponding to the IGEM distribution kit, they were sent for synthesis. Specifically, they were sent to IDT (Integrated DNA technologies) for synthesis. After that, the successive transformations were successful.
Golden Gate L1 Assembly
For assembly on a single plasmid promoter, RBS, terminator and gene, the Golden Gate technique was used. Firstly, the protocol followed indicated the successive application of cycles in the thermal cycler consisting of a very long incubation stage at 37ÂșC. This first attempt did not achieve the desired result. Therefore, different parameters were applied in the thermal cycler.
We proceeded to construct what is referred to as "Level 0" in the Golden Gate methodology. This term denotes each plasmid with an individual component. Subsequently, we continued to build the "Level 1" components. To accomplish this, we employed four distinct "Level 1" components, which consisted of four unique "Level 1" backbones denoted as A, B, C, and D. The differentiating factor was that each one was prepared to be employed in a subsequent step for the purpose of joining components in tandem. Although these components were identical, the region following enzyme cleavage was complementary, enabling the fusion of fragments.
These four backbones were sourced from the iGEM Kit and exhibited resistance to kanamycin, appearing green. We conducted enzyme digestions and selected colonies that lacked the green coloration. This selection criterion was due to the fact that the presence of the green color in the vector implied that the only means by which our construction could enter was contingent upon the departure of the green component and the subsequent introduction of our construct.
At this juncture, we identified positive results for 2 of the constructs, specifically, TAL and 4CL. In essence, we successfully isolated 2 genes.
At the end of that stage, we would have four Level 1 plasmids (A, B, C, D). Each of these plasmids would contain a complete construct comprising a promoter, RBS gene, and terminator for each of the genes: TAL, 4CL, and the red protein.
The next step is to utilize a Level 2 backbone, which we also obtained from the plate and analyzed. This backbone is identical to the Level 1 used previously, but it differs in antibiotic resistance. In this case, spectinomycin is the antibiotic resistance marker associated with Level 2. We used only one of these Level 2 backbones because the goal is to take the assemblies of our four Level 1 constructs and merge them in tandem, creating a single unit joined to the Level 2 backbone.
By doing this, we would achieve a complete Level 2 construct and four building blocks that we constructed in Level 1, each with its own promoter and terminator. This is feasible because Level 1A is compatible with Level 1B, Level 1C, and Level 1D, forming a part of Level 2. This allows us to complete the circle.
2. Revaluation of wine by-products
In order to get the wine industry waste byproducts ready to be used by our E Coli as substrate to produce resveratrol we could have gone with 3 different sample preparation methods.
Extraction-Purification
This method consists of making an acid hidrĂłlisis in the sample to separate the P-coumaric acid from the tartaric one. Then we should use a variety of chemistry techniques to isolate it and some others to extract it. We didn't choose this pathway because it didn't fit our idea of a circular economy, the reasons are it would be difficult to implement it in the wine industry, itâs also an expensive method because and there isnât much p-coumaric acid in this wine waste byproducts, in short, itâs not profitable.
Hydrolysis
The second way we think to proceed was just doing the hydrolysis in order to make the p-coumaric accessible to the E coli so it can be used to produce resveratrol, it's just a waste of time and money purifying the P-coumaric acid we thought, because E-coli should be able to use this precursor thatâs free on the substrate. But the downsides are that many other phenolic compounds would also be free and these ones are bactericidal so they could kill our E. coli.
Raw material
The last option, and the one we chose to make it happen, is trying to use the sample itself without any complex treatment and see if E.coli is capable of getting the p-coumaric acid or not. This would be affordable and easy to implement in any kind of industry, just a perfect example of a circular economy.
First of all we weight the amount of sample we want to work with, keeping in mind that when the process is finished we can end up with a half of the weight of the grape marc we started with. Then we have to dehydrate the sample, this is the most time consuming stage, with a 30ÂșC oven we have to spread the grape marc in a homogeneous thin layer so it dehydrates faster and equally.
The temperature is important to be low as we mentioned so we don't degrade any compound we have.
We will continue with the process when we have completed the dehydration. In order to know this we will have to weigh out the samples repeatedly. When the sample weight is constant for three consecutive days we take it out of the oven because it means it's fully dehydrated.
Then we grind it with a coffee grinder and proceed to sieve it with a 300 micrometer sieve.
Finally we saved it in a plastic container.
3. Bacterial growth model in the presence of sieve wine by-products
We have performed a bacterial growth test of BL21 E. coli bacteria (the ones we would used to biosynthesise resveratrol) with different concentrations of the extracted sieved wine by-products that we plan to use to boost the bioproduction of resveratrol once the final construction has been obtained. Growth was measured using a 24 well microplate with continuous measurements every 30 min for 24 hours at 660 nm.
The by-products were treated with 96Âș ethanol, crushed with a mortar and filtered after centrifugation. In this way, an extract corresponding to 0.05 grams/ml ethanol was obtained, which was added to each well at varying concentrations together with LB culture medium and BL21 bacteria.
The treatment added per well is as follows:
| Control | Extract | Ethanol 96Âș | |||
|---|---|---|---|---|---|
| Blank LB | Blank 0.1% | Blank 1% | Blank 5% | Blank EtOH 200 ”L | Blank EtOH 40 ”L |
| Control 1 | 0.1% 1 | 1% 1 | 5% 1 | 1 | 1 |
| Control 2 | 0.1% 1 | 1% 2 | 5% 2 | 2 | 2 |
| Control 3 | 0.1% | 1% 3 | 5% 3 | 3 | 3 |
For each treatment there were three replicates as shown in the table above. Growth was analysed in 0.1%, 1% and 5% extract. Since the extract is embedded in ethanol, and we wanted to analyse exclusively the impact of by-products, the last two columns of the microplates contained ethanol with the same volume as in the lanes at 1 and 5% extract. Also, the three control replicates contained exclusively LB + bacteria, while the blanks contained no bacteria.
In "Results", the data and graphs obtained, as well as the conclusions we reached, is available. Also, the following protocol is available in âProtocolsâ.