Results

In this section, the results of the cloning of the corresponding constructions carried out are presented: L0 and L1. The results obtained will be analyzed and future implementations (L2) will be discussed.

1. L0 cloning

pUC19 L0 cloning

The plasmid vector for the seven L0 constructs was pUC19. First, the plasmid was cloned individually into the bacteria. The efficiency of transformation was \(2,48 * 10^{7}\). Two minipreps (tubes 1 and 2) were performed, after which the resulting plasmid concentration was analyzed using Nanodrop. Tube 1 consisted of a lower and limiting concentration of 38.40 ng/ul; whereas tube 2 was higher and equal to 77.70 ng/ul.

Table 1. pUC19 miniprep concentration.
pUC19 miniprep Concentration (ng/µl)
Tube 1 38,40
Tube 2 77,70

An enzymatic digestion was performed with SmaI, and an electrophoresis of the digests and the miniprep plasmids was run. The digestion was confirmed as well as performed, obtaining bands close to 3000 bp.

Figure 1
Figure 1. pUC19 cloning electrophoresis. Two different transformations were analyzed. A miniprep and digestion were carried out, followed by electrophoresis. Lanes 1 and 3 correspond to miniprep plasmid, and lanes 2 and 4 to SmaI miniprep digestion. pUC19 is 2686 bp length. 1kb molecular weight marker was used.

TAL gene L0 cloning

Ligation of each of the parts to the pUC19 plasmid was performed independently but simultaneously. The results obtained were as follows:

The first construct to be obtained was PUC19_TAL. An initial transformation with the three genes STS, 4CL and TAL resulted in many TAL-positive colonies, but not the rest of the genes. Therefore, the first clone obtained was the one mentioned above. A PCR of 11 TAL colonies was prepared and electrophoresis was performed. Four positive colonies were obtained.

Figure 2
Figure 2. TAL L0 cloning electrophoresis. Fourteen colonies (11 TAL and 3 STS) transformed with pUC19 ligation to both genes were analyzed. Only four positive bands were obtained, corresponding to TAL, close to 1700 bp. A 1kb molecular weight marker was used.

STS gene L0 cloning

The next construction obtained was pUC19_STS. For this, positive transformed colonies were obtained with a ligation of STS and pUC19, and at the same time colonies with 4CL and pUC19 ligation were obtained. The latter were negative. As in the previous case, 15 colonies were taken and a PCR was prepared, which was then analyzed by electrophoresis. Two positive lanes (STS only) were obtained.

Figure 3
Figure 3. STS L0 cloning electrophoresis. Fifteen colonies (transformed with pUC19 ligation to both) genes were analyzed. Only two positive bands were obtained, corresponding to STS and lanes 7,10. A 1kb molecular weight marker was used.

4CL L0 cloning

The last L0 of the top three genes was pUC19_4CL. After unsuccessful attempts, many positive colonies were obtained, so PCR and electrophoresis of 16 colonies was performed. Many positive results were obtained.

Figure 4
Figure 4. 4CL L0 cloning electrophoresis. Sixteen (transformed with pUC19 ligation) were analyzed. Lots of positive bands gere obtained (1,2,3,4,5,6,7,8,10,12,13,14). A 1kb molecular weight marker was used.

pUC19_STS, pUC19_4CL and pUC19_TAL check

To make a final check that the three L0 plasmids had been obtained, we performed miniprep, digestion and electrophoresis from liquid cultures we prepared with the three genes.

The lanes analyzed each contain one:

Table 2. STS, 4CL and TAL L0 electrophoresis check
Lane DNA Expected size (bp)
1 pUC19_TAL miniprep
2 pUC19_TAL digestion 3876+526
3 pUC19_STS miniprep
4 pUC19_STS digestion 3374+520
5 pUC19_4CL miniprep
6 pUC19_4CL digestion 3490+758
Figure 5
Figure 5. STS, 4CL and TAL check electrophoresis. The digestion was carried out using the BamHI enzyme. The expected sizes were correct. A 1kb molecular weight marker was used

RBS, promoter, terminator and ppor L0 cloning

We had a lot of problems with the recovery of the previous iGEM kit parts, all of which had ampicillin resistance. Therefore, they were sent for synthesis, and these were the ones used. Ligation with the pUC19 vector and subsequent transformation was performed. Thirty colonies were then taken from the four transformed parts and electrophoresis was performed.

Figure 6.1 Figure 6.2
Figure 6. bs, promoter, terminator and ppor gene L0 clonation electrophoresis. As seen, lots of positives were found (of four parts). A 1kb molecular weight marker was used.

With the above clonings we can confirm that all ordered L0s were obtained correctly.

2. L1 cloning

pJUM29 L1 backbone cloning

The next step was to obtain and transform the plasmid we used for the four L1 constructs: pJUM29. They were recovered from the IGEM kit and transformed into bacteria, after which miniprep was performed and the concentration was measured.

Table 3. Concentration after transformation of pJUM29.
Part Concentration (ng/µl)
Backbone L1 A 22,35
Backbone L1 B 27,45
Backbone L1 C 31,80
Backbone L1 D 22,60

The data obtained were rather poor, although we still performed electrophoresis to confirm relative sizes. We observed that there was indeed plasmid.

Figure 7
Figure 7. Electrophoresis of four L1 backbone minipreps. Although the concentration measured was low in all four cases, we were able to confirm the existence of the plasmid.

Subsequently, we performed digestion and electrophoresis of the four backbones.

Figure 8
Figure 8. Check electrophoresis of four L1 backbone. Except for lane 4, electrophoresis allowed us to confirm that the cloning was correct. The expected sizes were 3000+90 bp. The molecular weight marker used was 1kb.

TAL and 4CL L1 cloning

The experimental part continued with the L1 cloning of the four genes STS, 4CL, TAL and PPOR. The first two genes to be cloned were 4CL and TAL.

Figure 9
Figure 9. Check electrophoresis of four l1 constructions. Only lane 1 (TAL) and lane 5 (4CL) were positive. Expected sizes were 1930+1844 and 2930+2001 respectively. The molecular weight marker used was 1kb.

PPOR L1 cloning

The next cloning achieved was for the PPOR gene. It was attempted together with the STS clone, which was negative.

Figure 10
Figure 10. Check electrophoresis of STS and PPOR constructions. Only lane 4 (PPOR) is positive. The molecular weight marker used was 1kb.

3. L2 cloning

pJUM49 L2 backbone cloning

For the L2 construction, the first obtained was the plasmid that acts as a backbone (pJUM49).

Figure 12
Figure 12. Check electrophoresis of pJUM49. Lane 7 corresponds to pJUMP49, and has a positive band. . The molecular weight marker used was 1kb

4. Weights of Pomace

The following table displays the different weighings of the fermented and unfermented pomace obtained after several days of drying and sieving. The aim of this technique is the complete drying of the pomace so that it does not contain any water and its subsequent sieving. In other words, separating it into fine and coarse particles in order to obtain p-coumaric acid.

Table 4. Weighing data for both types of pomace after several days of processing
Fermented pomaced (g) Unfermented pomace (g)
Day 1 155,44 200,6
Day 2 83,97 99,74
Day 3 83,97 99,82
Day 7 83,7 99,2
Day 8 83,43 99,2
Day 14 82,8 98,8
Day 15 82,89 97,01
Figure 11
Figure 13.Graphical representation of the weight values obtained for each type of pomace over time.

As can be seen in the graphical representation in Figure 13, a significant decrease in weight was observed after the first day of treatment in both samples. Thereafter, the decrease slowed dramatically and remained almost stable over the two weeks. In addition, the unfermented pomace maintained a higher weight than the fermented pomace throughout the fifteen days of drying and sieving. It could also be observed that the initial decrease in weight between the first and second day was more pronounced in the unfermented sample. In this way, the weight of the pomace samples (fermented and unfermented) can be determined after dehydration and sieving, in order to obtain the p-coumaric acid and use it for the synthesis of resveratrol.

5.Bacterial growth model in the presence of sieved wine by-products

Six different bacterial growth assays were performed:

  • Control:Bacterial growth in LB liquid medium.
  • Extract of wine by-products in 0.1% ethanol (4 microlitres).
  • Extract of wine by-products in 1% ethanol (40 microlitres).
  • Extract of wine by-products in 5% ethanol (200 microlitres).
  • Ethanol (40 microlitres).
  • Ethanol (200 microlitres).

A more detailed description of the experiment can be found in "experiments". The graphs obtained were the following:

Figure 14
Figure 14.Graphical representation of the bacterial growth model.

The growth control was correct, reaching the plateau phase at around eight hours of growth. Growth was almost identical and correct in the wells with 0.1% extract, while growth inhibition was observed in the wells with higher extract concentration. Finally, as expected, no growth was found in the wells with ethanol.

With this test it is possible to draw conclusions about the concentration of extract that could be used for resveratrol biosynthesis. Thus, the most appropriate given these results would be 0.1%. Mathematically, we can calculate the growth rate in the control and 0.1% wells, and observe that the differences are insignificant.

\[ln N = ln N_{0}+μΔt\]

\[μ = \frac{ln \frac{N}{N_{0}}}{Δt}\]

Control

\[N_{0} = 0.094666667\]

\[N = 1.12167\]

\[Δt = 7 hours\]

\[μ = \frac{ln \frac{N}{N_{0}}}{Δt} = \frac{ln (1.12167)/0.2597}{4 hours} = 0.3657 h^{-1}\]

0,1%

\[N_{0} = 0.135666667\]

\[N = 1.079666667\]

\[Δt = 7 hours\]

\[μ = \frac{ln \frac{N}{N_{0}}}{Δt} = \frac{ln (1.079666667)/0.2763}{4 hours} = 0.3407 h^{-1}\]

With these data we can conclude that the variation in the specific growth rate is small, so that the addition of 0.1% extract does not significantly change the growth rate of the bacteria, and therefore could be appropriate for bioproduction.

6. Summary and further development

In conclusion, as described above, all L0 and L1 clones were obtained. That is, all individual fragments (TAL, STS, 4CL, PPOR, RBS, prom and ter) were first successfully inserted into their respective ampicillin-resistant pUC19 plasmids. Then, all four L1 level clones were also obtained. Thus, we were able to obtain the Golden Gate constructs with the pJUMP29 plasmid corresponding to TAL, 4CL, PPOR and STS. In addition, parallel drying and sieving of fermented and unfermented pomace samples was also carried out to obtain the precursor p-coumaric acid.

Therefore, our next goal is to continue the laboratory work, in order to reach the final L2 construct. First of all, the L1 STS construct should be verified by performing an individual PCR of the six samples in the lane with the positive band. Then, the four L1 constructs (T7-LacO promoter, RBS, gene and T7 terminator) would be assembled in tandem in a pJUMP49 plasmid. Once the L2 clone has been successfully obtained, the bacteria should be able to express the genes and synthesize resveratrol from by-products of the wine industry. This ability could be assessed by supplying the dried and sieved by-products to the bacteria and analyzing resveratrol synthesis. Fermented and non-fermented samples will allow the evaluation of the effect of fermentation on resveratrol synthesis.

Looking to the future, we like to contemplate the possibility of encapsulating the synthesized resveratrol to introduce it into the commercial world. In this way, nano-encapsulated resveratrol could be offered to the general public and enterprises, with all the guarantees of protection, efficacy and stability. We would also be thrilled if another iGEM team would use our idea to move forward and bring resveratrol to the world .