iGEM 2023 — Team upnavarra-spain


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## Engineering success

![ Metabolic engineering of resveratrol production](https://static.igem.wiki/teams/4671/wiki/ruta-ingenier-a.png)

Figure 1. Synthesis pathway of resveratrol.

*Table 1. The three key vid enzymes for resveratrol synthesis.*

| Step | Precursor(s) | Products | Enzyme | Enzyme origin |
| 1 | Tyrosine | P-coumaric acid | TAL | From plants,bacteria or yeast |
| 2 | P-coumaric acid | P-Comaril-CoA | 4CL | From plants |
| 3 | P-Comaril-CoA  Malonyl-CoA | Resveratrol | STS | From plants |

As shown in Figure 1, the metabolic pathway of resveratrol synthesis from glucose involves many enzymes and
steps. However, *E.coli* is known to have the ability to produce tyrosine from glucose. Therefore, by
cloning the
relevant genes from grapevine, it could be enabled to produce resveratrol from tyrosine. For this reason,
cloning of the TAL, 4CL and STS genes, described in Table 1, will be performed in *E.coli*. Consequently,
our
*E.coli* will reproduce the metabolic pathway and will be able to synthesize resveratrol from p-coumaric
acid, a
precursor found in wine by-products.

However, in order to maximize the production of resveratrol from wine industry waste by-products, all possible
precursors have to be considered. As also shown in Figure 1, there are two main precursors of resveratrol:
p-Coumaroyl-CoA (from p-coumaric acid) and malonyl-CoA. Malonyl-CoA is a key molecule in the fatty acid
biosynthesis pathway and is rapidly consumed in the cell, so it is found in reduced amounts, limiting
resveratrol production. Therefore, another plasmid encoding for the enzyme ScACC (this enzyme converts
acetyl-CoA to malonyl-CoA) could be constructed to increase malonyl-CoA concentrations in the cell.

Overall, our main goal is to achieve the production of synthetic bacteria with two plasmids of interest: one
vector with the grapevine genes and another one for enhanced malonil-CoA production. The resulting synthetic
bacteria will be able to increase resveratrol production from p-coumaric acid and malonil-CoA, both precursors
obtained from by-products of the wine industry waste.

## The Golden Gate Cloning Method

The Golden Gate technique is a cloning method that allows the assembly of multiple DNA fragments in a
user-defined arrangement. This technique utilizes type IIS enzymes. Firstly, the recognition site of the enzyme
is non-palindromic (between 4 and 7 nucleotides). Additionally, its cutting sequence is outside the enzyme's
recognition sequence, making the assembly functionally scarless.

Various type IIS enzymes are used to cut DNA and produce different ends. For example, the enzyme we use, BsaI,
leaves a 4-nucleotide overhang on the strand. By making the cut outside the enzyme's recognition site, the ends,
also known as fusion sites, can be designed for compatibility between parts. BsaI can generate 256 possible
combinations of nucleotides for fusion sites.

![Golden gate 1](https://static.igem.wiki/teams/4671/wiki/goldengate-1.png)

Blue: restriction enzyme recognition site.

![Golden gate 2](https://static.igem.wiki/teams/4671/wiki/goldengate-2.png)

The orientation of the recognition sites of IIS enzymes and fusion sites is critical for the success of the
Golden Gate technique. In the target vector, the sites must be facing away from each other. As a result of
cutting after the recognition sequence, the remaining ends are not complementary to each other, preventing the
vector from closing once the IIS enzyme is added.

![Golden gate 3](https://static.igem.wiki/teams/4671/wiki/goldengate-3.png)

## The engineering cycle

In our project, one engineering cycle has been designed, divided into levels L0, L1 and L2. The design - build
- test - learn cycle was developed in the laboratory, based on the set of constructs shown below.

At the L0 level, the four genes of interest (STS, 4CL, TAL and PPOR) and the other three fragments (T7-LacO
promoter, ribosome binding region or RBS, and T7 terminator) must be synthesized individually. The PPOR gene
encodes a red fluorescent protein that will allow us to confirm the correct expression of the other three genes,
due PPOR has the same elements as the other constructions. Thus, each fragment is introduced into a pUC19
plasmid with ampicillin resistance using the restriction enzyme SmaI (blunt ends), resulting in seven L0
plasmids.

![pUC19 plasmid (2686 bp)](https://static.igem.wiki/teams/4671/wiki/cycle-1-def.png)

Figure 2. pUC19 plasmid (2686 bp)

![Level 0 construction method](https://static.igem.wiki/teams/4671/wiki/cycle-2-def-2.png)

Figure 3. Level 0 construction method

At the L1 level, four Golden Gate constructs are made, one for each gene of interest (STS, 4CL, TAL and PPOR).
Each kanamycin-resistant backbone (A, B, C and D), obtained from the iGEM kit, contains a full construct with a
T7-LacO promoter, a RBS and a T7 terminator for each gene, to ensure correct gene expression. To obtain L1
constructs, we employed pJUMP29 as backbone. Both the backbone and each L0 vector are digested with BsaI enzyme
and ligated, generating the correct construction.

![Level 1 construction method](https://static.igem.wiki/teams/4671/wiki/cycle-3-def-2.png)

Figure 4. Level 1 construction method

![Constructions generated in this work](https://static.igem.wiki/teams/4671/wiki/cycle-4.png)

Figure 5. Constructions generated in this work. (A) pJUMP-1A-construction,
(B) pJUMP-1B-construction, (C) pJUMP-1C-construction, (D) pJUMP-1D-construction

At the L2 level, a single spectinomycin-resistant construct must be elaborated using the pJUMP49 plasmid,
containing all four genes. Each L1 backbone construct (T7-LacO promoter, RBS, gene and T7 terminator) is
extracted and then assembled in tandem, resulting in an L2 plasmid with all four constructs. This Golden Gate
construct is assembled using the BsmBI enzyme.

![Level 2 construction method](https://static.igem.wiki/teams/4671/wiki/cycle-5-def-2.png)

Figure 6. Level 2 construction method, being “A” the TAL transcriptional
unit, “B” the 4CL transcriptional unit, “C” the STS transcriptional unit and “D” the PPOR transcriptional
unit, each of them containing the promoter, RBS and terminator sequence.

![pJUMP level 2](https://static.igem.wiki/teams/4671/wiki/cycle-6.png)

Figure 7. pJUMP level 2

Thus, this final 9217 pb construct would enable the bacteria to express the genes and synthesize
resveratrol from by-products of the wine industry (Figure 7). However, since the T7-LacO promoter is
inducible by IPTG, it would be necessary to remove glucose and provide lactose (or IPTG) to the bacterial
culture, in order to achieve a better gene expression.

## Fine-tuning of engineered bacteria

Once the engineered bacteria have been successfully obtained, an analysis of bacterial growth can proceed.
Bacterial growth can be recorded according to the concentration of sieved by-products supplied. For this,
different known sieve concentrations can be determined and fed to our engineered *E.coli*, which must be monitored
periodically (e.g. every 1h) for a specific period of time. This will allow the respective growth curves to be
produced. From this data it will be possible to determine the sieving concentration at which the bacteria have
an optimal growth, and therefore a better resveratrol synthesis.

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## 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):

[![Golden Gate process](https://static.igem.wiki/teams/4671/wiki/goldengate.png)](https://www.snapgene.com/guides/golden-gate-assembly)

[Figure 1.
Photograph of Golden Gate assembly. (s/f). Snapgene.com.](https://www.snapgene.com/guides/golden-gate-assembly)

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 37ºC. 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 hidrólisis 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,
it’s also an expensive method because and there isn’t much p-coumaric acid in this wine waste byproducts, in
short, it’s 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 that’s 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 30ºC 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.

![](https://static.igem.wiki/teams/4671/wiki/molinillo.jpg)
![](https://static.igem.wiki/teams/4671/wiki/tamiz.jpg )

Finally we saved it in a plastic container.

![Lab photo](https://static.igem.wiki/teams/4671/wiki/guardado-orujo-tamizado.jpg)

## 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 96º 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 96º | |
| --- | --- | --- | --- | --- | --- |
| Blank LB | Blank 0.1% | Blank 1% | Blank 5% | Blank EtOH 200 µL | Blank EtOH 40 µL |
| 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 *“Protocols”*.

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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](https://static.igem.wiki/teams/4671/wiki/figure-1.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-2.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-3.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-4.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-5.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-6-1.png)
![Figure 6.2](https://static.igem.wiki/teams/4671/wiki/figure-6-2.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-7.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-8.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-9.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-10.png)

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](https://static.igem.wiki/teams/4671/wiki/figure12b.png)

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](https://static.igem.wiki/teams/4671/wiki/figure-12.png)

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](https://static.igem.wiki/teams/4671/wiki/figure14.png)

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 .

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