Day 1. July 17th

Today we started the experimental part of the project in our laboratory. First, we reviewed the safety standards in the laboratory and the resources available to carry out our work. At first, we planned the steps to follow during this week. Our first goal is to make our cells competent by heat shock.

During this day the preparation of solid media Agar LB was carried out. Liquid medium already prepared was melted and other LB Agar media were prepared if necessary. Finally, the cells were inoculated in petri dishes with the medium and subsequently left to grow at 37ºC.

Day 2. July 19th

Our objective on this day was to obtain competent cells from the culture started on day 17. After a few hours of culture in a liquid medium at 26ºC, we met to obtain competent cells according to the protocol. However, the measurement of the OD of the cell culture gave us a value of \(0.110\); well below the appropriate values \([0.4;0.6]\). Therefore, we increased the temperature to 32ºC and left the cells shaking for approximately 50 minutes. After the said time, the OD was \(0.244\).

Although we tried to continue with the protocol despite this, we finally decided to restart the liquid culture. We took cells from the petri dishes and started the liquid culture again at 37ºC. Finally, we decided to meet again on the 21st to obtain the competent cells.

Day 3. July 21th

This day we went to the laboratory in the morning to try to get the competent cells. The bacteria had been shaken cultured overnight at 20°C. The first measurement of OD gave us a value of \(0.157\); a value much lower than desired, so we increased the temperature to 26ºC and waited for approximately one hour. During that time, we reviewed the gene constructs.

When we got back to the lab, the OD was \(0.381\), so they were ready to continue with the protocol. Following the corresponding steps, we obtained a total of 42 tubes with prepared cells. Finally, we stored them at -80ºC. We agreed to meet in the lab next July 26.

Day 4. July 26th

Our objective on this day was to carry out the transformation of our competent cells to introduce the pUC19 plasmid with resistance to ampicillin into them. The plasmid concentration was \(10 pg/ug\). We did the transformation by heat shock at 37ºC, which we performed even though the protocol did not express it as necessary. Finally, we obtained petri dishes with ampicillin resistance through serial dilutions. The plates were stored at 37ºC for around 24 hours. We agreed to meet the next day to note the efficiency of the transformation.

Day 5. July 27th

During this day, we checked the efficiency of the bacterial transformation of the previous day. The five plates obtained during the previous day have grown correctly. Given that all of them had the antibiotic ampicillin, and that it was the pUC19 plasmid that conferred resistance to the bacteria, we can ensure that the insertion of the plasmid was correct. Next, we took the most diluted plate and transferred some colonies to another plate, which we incubated for a few hours at 37ºC.

In addition, we measured the efficiency of the transformation with that same board, which turned out to be very good. We agreed to meet the next day.

\[Efficiency = {70000000 CFU* \frac{1}{10 pg ADN}*\frac{1000 pg}{1 ng}*\frac{1000}{ug}= 7*10^{12}\frac{CFU}{ug DNA}}\]

Day 6. July 28th

Our objective today was to carry out an electrophoresis of the plasmid inserted in our bacteria, and to carry out an enzymatic digestion in order to prepare our inserts (the genes). However, as soon as we arrived at the laboratory we ran into a setback. Our secondary PI (Lucas Castellani), who is guiding us through the work in the laboratory, told us that he had incubated the strain of bacteria we are using without the pUC19 plasmid on an ampicillin plate. The surprise was that many colonies had grown, which told us that somehow the strain of bacteria we are using has natural resistance to ampicillin. Therefore, the cloning of the previous day most likely had not occurred. And that is why the efficiency was too high.

Likewise, to clear up doubts, we performed a miniprep and the corresponding electrophoresis of four samples: two corresponding to our supposedly transformed bacteria, and another two control samples. As we suspected, our colonies did not have any inserted plasmids, and indeed the strain we used had spontaneous resistance to the antibiotic.

We agreed to wait for a few days for the growth of two plates that we have prepared: one with the same concentration of ampicillin, and another with twice as much; with the aim of studying whether an increase in the concentration of the antibiotic is not naturally tolerable by the strain.

Day 7. July 29th

Our PI Lucas Castellani warned us that different strains of E. coli had grown in media with ampicillin, so we could be facing a problem with this antibiotic. We agreed to wait a few days to see if it was possible to solve the problem before continuing with the work in the lab.

Day 8. August 1st

Our PI Lucas Castellani told us that after testing a new stock of ampicillin, the bacteria did not grow in the presence of the antibiotic. Therefore, we could now continue with the work in the laboratory. We agreed to return to the lab the next day

Day 9. August 2nd

Already with the correct antibiotic, today the objective was to transform the bacteria again following the same protocol as on July 26. In addition, we made an extra plate, which will serve as a control to ensure that the antibiotic works correctly.

Day 10. August 3rd

Just like we did on July 27, today we had to measure the efficiency of the transformation. The results have been visibly better than those of the other day, by obtaining an average efficiency data much closer to what should come out. The three plates looked as follows: the plain plate had approximately 121 colonies, while the \(10^{-1}\) dilution had a total of 8 colonies. The \(10^{-2}\) dilution plate had a single colony. We take colonies to put them in liquid culture, so the next day we can do the miniprep.

\[Efficiency = {1241 CFU* \frac{1}{50 pg ADN}*\frac{1000 pg}{1 ng}*\frac{1000 ng}{ug}= 2.48*10^{7}\frac{CFU}{ug DNA}}\]

Day 11. August 4th

Today we had to carry out the miniprep of the two liquid cultures taken from the transformed bacteria. To do this, we used the same procedure as on July 28. We then prepared an enzymatic digest of the plasmid using the Smal enzyme. We measured the DNA concentration, and for each tube the results were:

• Tube 1: \(38.40\) ng/uL
• Tube 2: \(77.70\) ng/uL

Enzymatic digestion was done by adding the following components:

	Volume (Tube 1) (uL)	Volume (Tube 2) (uL)
Buffer	3	3
Plasmid DNA	16	8
Smal enzyme	2	2
Water	9	17
TOTAL	30	30

Some hours later, our PI Lucas Castellani carried out an electrophoresis from which we will continue lab work on August 7th. This was the result

Day 12. August 7th

Today's objective was to perform the ligations of the three genes: TAL, STS and 4CL to the pUC19 plasmid that we already digested the day before. We used the plasmid from tube 1. This is so because the electrophoresis on day 4 showed a higher concentration of DNA in the digestion of tube 1 than in tube 2.

For each gane we used the following:

	Volume (uL)
Buffer	2
Plasmid DNA	2.5
Ligase	0.5
Insert DNA (gene)	5.0

Plasmid pUC19 was heat inactivated at 65°C for the STS gene mix. This is so to avoid unnecessary cuts in the gene. We agreed to meet on August 8 to carry out the transformation of the ligations in bacteria.

Day 13. August 8th

The objective today was to carry out the bacterial transformation of the three ligations committed yesterday. To do this, first of all, we have performed three transformations in three eppendorfs tubes. Each of them for a plasmid with its corresponding gene. While we let them incubate for an hour, we prepared six petri dishes with LB medium, ampicillin and Xgal. The Xgal will allow us to detect those colonies that have possibly taken the genes linked to their plasmid. Three of these plates were extra to use at another time, although solid clumps of LB formed on them. The other three, the ones we needed for each of the ligations, were plated correctly.

Correspondence of LB-antibiotic-Xgal volumes:

	Volume
LB medium	50 mL
Xgal	100 uL
Ampicilin 200X	25 uL

Finally, we added the corresponding transformed bacteria to each plate. Therefore, we have left three plates to incubate for one day at 37ºC: one for 4CL, another for TAL, and another for STS.

Day 14. August 9th

Our objective today was to review the three plates from yesterday in order to continue with our project. The plate with bacteria with the TAL gene had white and blue colonies, so it had turned out well and we could continue with the DNA analysis of the white colonies.

the other hand, the plates with bacteria with 4CL and STS had very few white colonies (STS none), so the transformation of both genes had not been achieved correctly.

Plate	Number of white colonies
TAL	19
4CL	0; Blue colonies only
STS	3

We proceeded with the STS and TAL plates, from which we took 14 colonies for PCR (3 STS colonies and 11 TAL). In addition, we included 2 more tubes as controls: one with only water (negative control) and another with a blue colony. In total, 16 PCR tubes in which we included the following premix:

TOTAL	100 uL
5x Buffer	20 uL
dNTPs (2.5 mmM each)	8 uL
Fw	5 uL
Rv	5 uL
Mg (25 mM)	12 uL
H20	49.5 uL
Taq	0.5 uL

The PCR programme we used was the following:

In addition, two new plates were made with LB, Ampicillin and Xgal to re-obtain colonies of bacteria that have inserted the STS and TAL genes. We agreed to continue the next day with the results of the PCR and the new colonies.

Day 26. September 6th

Previously transformed plates did not obtain colonies. Given the difficulties we are having, we have ordered a synthesis of the parts. Today, we have transformed the ligations of these parts. Four plates have been left to grow, hoping to obtain colonies tomorrow.

Day 27. September 7th

Today we have finally obtained colonies on plates transformed yesterday. As seen in the images, all four have white and blue colonies, although two of them have too much cell density.

We have performed a PCR of 30 tubes (7 colonies per plate and two negative controls). Subsequently, two electrophoresis were performed, each with 14 colonies from two plates. The results were the following:

Both results allow us to conclude that we have cells transformed with the plasmids of the four parts.

Day 28. September 8th

The objective today was to prepare the mix of the four L1 constructions of Golden Gate. To do this, first, a miniprep of the ampicillin parts and its corresponding electrophoresis were carried out.

Day 29. September 11th

During this day, we carried out the bacterial transformation of the four Golden Gate mixes prepared the previous day. In four plates, we let them incubate for 24 hours in the oven.

Day 30. September 12th

During this day, first of all we review yesterday's plates. The colonies obtained were all green (negative), so we had to repeat the four Golden Gate mixes, which were left in the thermocycler.

Day 31. September 13th

As we did on day 11, we performed the transformation of the four plasmids again, as well as the corresponding growth plates.

Day 32. September 14th

Today we reviewed the plates grown yesterday. Fortunately, we had (few) non-fluorescent colonies, so we decided to continue. Since we could not find a way to obtain a specific enzymatic pattern for PCR analysis, we decided to streak a new plate and a liquid culture so that we could do a miniprep tomorrow.

Day 33. September 15th

Following what was done yesterday, today we did miniprep, digestion and electrophoresis of some colonies of each of the four L1 constructions. However, electrophoresis did not allow us to observe the expected fragment pattern for each sample. First, we made a mistake by using the wrong enzyme (NdeI instead of BgIII). Still, the observed pattern is atypical, as we observe a similar pattern in almost all of the digestions with this enzyme.

MW	Molecular weight marker	6	ppor 1 Digestion	12	STS 2 Digestion
1	TAL 1 Miniprep	7	ppor 2 Miniprep	13	4CL 1 Miniprep & 4CL1 Digestion * A mistake in the process of sample introduction led to the insertion of these two samples in the same well.
2	TAL 1 Digestion	8	ppor 2 Digestion
3	TAL 2 Miniprep	9	STS 1 Miniprep	14	Empty well
4	TAL 2 Digestion	10	STS 1 Digestion	15	4CL2 2 Miniprep
5	ppor 1 Miniprep	11	STS 2 Miniprep	16	4CL2 2 Digestion

Day 34. September 18th

The plate with the L2 transformation did not grow beyond a few small dots.

We performed a digestion with NotI, a fast digesting enzyme. The plasmids used in the digestion are: TAL1, TAL2, ppor1, STS1, 4CL. The volumes used are:

Buffer	1 ul
Plasmid	5 ul
Enzyme	0.5 ul
Water	3.5 ul
TOTAL	10 ul

The corresponding electrophoresis was prepared:

In addition, we did another digestion with BsaI for the four backbones, four plasmids and three parts. The plan, given the problems we are encountering, is to use another vector to do the Golden Gate construction. The volumes used for each part were:

Day 35. September 19th

Today we found that the L2 plates had grown unexpectedly and had many fluorescent colonies. We did miniprep and enzimatic digestion of the genes, as well as an electrophoresis to evaluate the presence of plasmid.

Day 36. September 20th

Today, we prepared five minipreps of the ppor gene, as well as one of the L2 stretch marks grown yesterday. After that, the corresponding electrophoresis was done:

The 5 lanes before the marker are ppor digestions, negative since the fragment is not released. The next street is the lvl2 backbone which is perfect, and the last 2 are the new analyzes of TAL, which is still a bit strange.

Day 37. September 21st

Our PI Lucas Castellani prepared yesterday plates with different enzymatic digestions of the ppor and STS genes. Today, we selected 48 non-fluorescent colonies from both plates, which were transferred to a new kanamycin plate.

During the afternoon, we took the streaks from the newly grown plate and prepared ten liquid cultures (five of each gene), which we left to shake.

Day 38. September 22nd

Today the plan was to perform a mini-prep and check the STS and ppor ligations. However, after doing the miniprep we forgot to do the digests and did the electrophoresis directly. Evidently, this is not useful for evaluation. We will try to come back to it next day.

Day 39. September 25th

Based on the minipreps performed the previous day, we prepared NotI digests for five STS and five ppor colonies. Unfortunately, subsequent electrophoresis did not find the desired pattern in any of them.

Day 40. September 26th

Today we again performed miniprep, digestion and electrophoresis of ten liquid cultures of STS and ppor. This time, we took as colonies that were used for Golden Gate. As a result, we obtained a ppor positive colony (the fourth one excluding markers on the photograph), with a desired band of around 1000bp. However, we still had no STS positives.

Day 41. September 27th

Like yesterday, we performed miniprep, digestions and electrophoresis again. This time from ten STS gene colonies (five of them from the previous Golden Gate ligation and another five transformed last week). Unfortunately, we still did not find the desired pattern (1500 bp) in any of them.

Day 42. September 28th

We performed miniprep, digestions and electrophoresis of ten new STS colonies. Unfortunately, we still did not find the desired pattern in any of them. For digestion we use the enzyme SmaI. STS only has one restriction site for SmaI, so digestion will only be carried out if we have the plasmid. As seen in the photograph, there is no digestion, so we still have no positives.

To finish the day in the laboratory, we performed an electrophoresis of an STS digestion with the pUC19 vector. The objective was to check if the pattern we have been looking for these days is really what we should expect or if, on the contrary, there is some restriction site that we are not taking into account. However, we have found no evidence that this is the case.

Day 43. September 29th

Given the problems we are having in finding L1 STS positive colonies, yesterday our PI Lucas Castellani prepared digests of the necessary L0s: rbs, promoter, terminator, gene and backbone. Today we have prepared an electrophoresis of these digests, and we have been able to find the problem: as it can be seen in the image, the pUC containing STS (fifth lane from the left) is not digesting with the enzyme we need.

We also started the substrate manipulation. In the afternoon we weighed 200,60 g of non fermented sample and 155,44 g of fermented sample and took it into the oven at 30ºC. (this weights are obtained by measuring the plastic container with the grape marcs and then subtracting the plastic container weight).

Day 44. October 2nd

Given Friday's result, we went through the STS L0 notes first thing today. As far as we knew, the STS L0 clones in August had done well. We checked our stored L0 reagents, found two STS tubes, and did an enzymatic digestion with BamHI to check their status. Subsequently, we performed an electrophoresis including also undigested plasmids. The results obtained are as follows, showing a candidate in lane 4, corresponding to one of the digests.

Day 45. October 3rd

Our PI Lucas Castellani recovered plates with L0-transformed bacteria, which were still alive. We were able to recover colonies with RBS, one with promoter, one with terminator and two with STS. We performed a PCR of the five cases. We did electrophoresis, adding two STS digests with BsaI.

With the exception of the molecular weight marker, the first lanes correspond to the rbs, terminator and promoter parts. In principle in line with expectations. Lanes 4 and 5 correspond to the STS of the PCR, one of them (5) with an expected pattern. Finally, the digests for some reason do not seem to have occurred.

We decided to do digests of the two L0s of the PCR and see how we continue tomorrow.

Day 46. October 4th

Today we started with electrophoresis of yesterday's digests (the STS L0s) + the plasmids of the PCR-amplified parts themselves. The difference in length between the digests and PCR of the STS genes (last four lanes not counting the marker) is 125 bp, which is hardly visible in the image. This is why we do not know if the digestion has actually taken place, although a slight difference in size seems to be observed in the last two lanes (one of the digests and the corresponding gene). We have taken the digestion and performed two digestions, one of them including the Kpn enzyme to better discriminate sizes.

We also made another weight of the waste samples, the fermented one weighed 83,97g and the non fermented one weighed 99,82g.

Day 47. October 5th

Today, we transformed yesterday's two STS alligations into two plates, which we left to grow in the oven at 37ºC. In addition, we performed a ligation to the two corresponding PCR products with BsII, and prepared an electrophoresis. The gel, seen at the bottom, was intended to confirm that the L0 cloning was correct. The expected sizes were 576+735, and the fragment found is the correct one.

Today we also measured the grape marcs, the fermented one weighs 83,97g and the non fermented one 99,74g.

Day 48. October 6th

Today we reviewed the growth of yesterday's plates. There are a lot of colonies, most of them non-fluorescent, which is strange because we have always found more fluorescent than non-fluorescent colonies. However, this is not necessarily a bad thing. We have streaked 30 colonies from both plates, and we will take a few hours later for liquid culture.

When we measured the grape marcs we saw today, the fermented one weighed 83,70g and the non one weighed 99,2g, so we decided to make a new measure on monday and see again if it keeps losing weight or if it keeps stable.

Day 49. October 7th

Today we performed miniprep, enzymatic digestion and electrophoresis of six liquid cultures transformed with STS L1. The enzyme used was SmaI and the result is shown in the image below: although there seems to be enzymatic digestion, two cut-off bands were expected instead of one.

Day 50. October 9th

Our initial aim on the present day was to check the expected digestion sizes and to perform further digests to the minipreps of the other day. However, when we reviewed the data we realized that we did not even have a pattern corresponding to the empty plasmid. So, we decided to redo the liquid cultures and continue with the checks tomorrow.

Day 51. October 10th

There are only two days left until Freeze day, so today we went back to miniprep, digestion and electrophoresis of six liquid cultures in order to try to finally get the L1 from STS. However, we have not been able to get positive results either. We think there may be problems with the L0 digestion that we are building on. We are awaiting primers that will allow us to do mass PCR screening of all the colonies.

On the other hand, after weighing the fermented and unfermented pomace for several days, it has been observed that the fermented pomace has reached a stable weight, while the unfermented pomace still varied. The fermented pomace has been grinded and sieved.

Day 52. October 11th

Last day before Freeze Day. Today a PCR was performed on thirty colonies in six tubes (five colonies in each), plus a positive control. The aim was to do a large screen to see if we found any L1 STS positive colonies. After PCR, electrophoresis was performed.

As can be seen, there is a faint band in lane four of the electrophoresis, and it coincides with the positive pattern. Pending confirmation, we have detected a correct cloning of the L1 STS.

So far our work in the lab before Freeze Day. However, we will continue our work in the lab over the next few weeks with the aim of trying to obtain the final construction (L2) and resveratrol quantification before the Jamboree.

The unfermented pomace has been reweighed, and it has been observed that it has reached a stable weight. It has been grounded and sieved.

Day 53. October 13th

Today we went back to the lab to perform the PCR check on pool number four where we found a positive last day. The five colonies that made up the pool were taken individually. After PCR, an electrophoresis was performed.

The image shows that no colony was found with L1 (on the left is a positive pattern). We found this very strange, since the previous day we had a very clear band with the correct pattern. We decided to run the PCR and electrophoresis again, this time with more colonies (from other pools) in case we had made a mistake when taking the samples.

Unfortunately, as shown in the picture below, the result was negative again. Next day we will review the notes and try to get results again.

Day 54. October 16th

As during the previous day we could not find the colony that was supposed to be L1 STS positive, today we performed an individual PCR on the 26 colonies from which we obtained the "positive" result. Unfortunately, we could not find the expected result. We assume, therefore, that the faint positive band the other day may have been due to contamination.

Day 55. October 17th

Given yesterday's results, we are back to square one with STS today. We did a miniprep and digestion of L0 7 and 10 with which we already worked a few days ago. We store the digestion ON at 37ºC.

Day 56. October 18th

Today we did electrophoresis of yesterday's digests, which came out rather strange. So, we prepared them again and left them overnight in the oven.

In addition, we have started to prepare for the bacterial growth study we are going to do. The idea is to use a strain of E. coli suitable for protein expression, called BL21, which would also be the final strain where we would synthesize the resveratrol. For the growth model, we will use the transformed strain with the L2 backbone that we already have. Today we have done that transformation and plating.

Day 57. October 19th

During the current day, first of all we checked yesterday's boards with the BL21 transformations of the L2 backbones, and they have grown correctly.

On the other hand, we have performed electrophoresis of the L0 digests of the STS, and the result confirms that the enzyme is not digesting the plasmid correctly.

The current strategy is to obtain the complete synthesis of the STS by PCR, so today we have obtained the necessary primers. After that, the result was positive, so a digestion was performed, which we will ligate tomorrow.

Day 58. October 20th

Today we performed the transformation of bacteria with the new L0 STS linked to the pJEM plasmid. In addition, we made three new STS L1 ligations with yesterday's PCR product, which we left ON at room temperature.

Day 59. October 21st

Today we checked the plate with yesterday's transformation, from which we only obtained three colonies. We have also performed the corresponding PCR. On the other hand, we have performed the transformation and plating of the three STS L1 ligations from yesterday.

Day 60. October 22nd

Today we went to the lab to perform PCR on the three STS plates from yesterday. Of the plates, only two colonies appeared to be non-fluorescent, so we did PCR on those two. We then performed an electrophoresis of this PCR and the ones we did yesterday, with the following results: one of the two L1 STS candidates was positive, and the rest of the L0 transformations were positive too. However, one of the negative controls was amplified, which is strange and will be checked tomorrow. We have prepared liquid cultures of the possible L1 in order to continue.

Day 61. October 23rd

During today we performed miniprep, digestion and electrophoresis of the liquid cultures we prepared yesterday that could contain a "positive" L1 STS. Unfortunately, the digestion did not work, and failed again.

On the other hand, we re-ran PCR of all transformations to check yesterday's result, in which there was a positive in the negative control. It turns out that the primers stick in the empty vector (even with 5 and 6 errors in the primers), on the resistance gene, and JUST amplify a size almost equal to what they amplify in our fragment (STS). We will keep trying!

Day 62. October 24th

Today we performed a new PCR with different primers on the three non-fluorescent colonies that we had not yet screened on the plates with the possible L1 STS transformations. In addition, we performed an enzymatic digestion of the pJEMT clone to check it. After that, we ran a gel. The electrophoresis showed that the pJEMT digestion was correct, so we performed a possible new ligation of L1 with this plasmid. At the same time, two of the colonies (on the left of the photo) seem to have tested positive, so we have prepared liquid cultures to check them tomorrow.

In addition, we have started the bacterial growth model. We have seeded various treatments in 24 wells and left them in the microplates for evaluation. Tomorrow we will collect the results.

Day 63. October 25th

Today we had two objectives: to perform a new transformation of the L1 STS linked to the pJEM-T plasmid; and to perform miniprep, digestion and electrophoresis of yesterday's liquid cultures. Again, the resulting electrophoresis was negative, so we will continue from tomorrow with the new pJEM-T plasmid, to see if we can increase the low efficiency we have been experiencing for weeks.

Day 64. October 26th

Today we performed PCR on 14 non-fluorescent colonies after yesterday's transformation. The electrophoresis was (again) negative for all colonies except the positive control.

We decided to make one last attempt using Golden Gate's own ligation cycling. Thus, we performed the ligation and left it for four hours in the thermal cycler. In the afternoon, after cycling, we performed the transformation into NEB Turbo cells and the corresponding plating.

Day 65. October 27th

Of the plates from yesterday's transformation, only 2 non-fluorescent colonies appeared. We did a PCR of both using three different primer combinations, so that we could see if there are any false positives. We were positive for one of the primer combinations, but not for the others. Although we assumed it was a false positive, we did a miniprep and digestion to remove any doubts.

The result after electrophoresis of the digestion was also negative. Given the short time available, we did not see the need for another ligation. We did a plating of the centrifugation of the same transformation as yesterday, in case anything grew.

Day 66. October 28th

Today was our last day in the lab, a very short one. Only one colony grew on the plate we left yesterday, and it was fluorescent. So this is the end of our journey through the experimental part of the project. Although we didn't get the last L1 or L2, we are very proud of what we have achieved, and we hope that this can be of help to future iGEM teams.