iGEM 2018 — Team Tartu_TUIT

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# Team:Tartu TUIT/Notebook

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* Project
  + [Description](/Team%3ATartu_TUIT/Description)
  + [Design](/Team%3ATartu_TUIT/Design)
  + [Modeling](/Team%3ATartu_TUIT/Model)
  + [Parts](/Team%3ATartu_TUIT/Parts)
  + [Results](/Team%3ATartu_TUIT/Results)
* Laboratory
  + [InterLab](/Team%3ATartu_TUIT/InterLab)
  + [Experiments](/Team%3ATartu_TUIT/Experiments)
  + [Notebook](/Team%3ATartu_TUIT/Notebook)
  + [Safety](/Team%3ATartu_TUIT/Safety)

* People
  + [Team](/Team%3ATartu_TUIT/Team)
  + [Attributions](/Team%3ATartu_TUIT/Attributions)
* Human Practices
  + [Human Practices](/Team%3ATartu_TUIT/Human_Practices)
  + [Public Engagement](/Team%3ATartu_TUIT/Public_Engagement)
  + [Collaborations](/Team%3ATartu_TUIT/Collaborations)

NOTEBOOK

Searched for the pathway of synthesis of mycosporine-like amino acids from the genes of different organisms. Actinosynnema mirum DSM with the gene names amir\_4256, amir\_4257, amir\_4258, amir\_4259. And Nostoc commune with the gene names mysA, mysB mysC and mysD.![](https://static.igem.org/mediawiki/2018/8/80/T--Tartu_TUIT--notebook1.jpg)

iGEM distribution kit arrived.![](https://static.igem.org/mediawiki/2018/4/41/T--Tartu_TUIT--notebook2.jpg)

Ordered synthetic DNA to initiate the lab work process.

Ordered primers for biobricks.

Primers arrived.![](https://static.igem.org/mediawiki/2018/d/dd/T--Tartu_TUIT--notebook3.jpg)

Started the first cloning process Biobricks with PCR, followed by restriction and overnight ligation.![](https://static.igem.org/mediawiki/2018/5/51/T--Tartu_TUIT--notebook4.jpg)

First bacterial transformation. Resulting colonies on most of the plates.

Obtained mysA biobrick sent for sequence determination. Positive result.

Interlab: we took two colonies from each of transofrmation plates and inoculated them in seperate tubes of 5 mL of LB+CAM.

Team photoshoot. ![](https://static.igem.org/mediawiki/2018/c/cc/T--Tartu_TUIT--notebook5.jpg)

CPEC, gybson, vegas cloning initiation. PCR with Phusion Polymerase.![](https://static.igem.org/mediawiki/2018/2/23/T--Tartu_TUIT--notebook6.jpg)

|  |  |
| --- | --- |
| H2O | 31.6 |
| 5xHF buffer | 10 || 25mM dNTP's (,,Template DNA) | 0.4 |
| Forward primer | 2.5 |
| Reverse primer | 2.5 |
| Template DNA | 1 |
| DM50 | 1.5 |
| Phusion polymerase | 0.5 |

-Skype conference with Ecuador team![](https://static.igem.org/mediawiki/2018/5/52/T--Tartu_TUIT--notebook7.jpg)

Successfully completed interlab. **�Achievement**- Fulfilled the bronze metal requirement.

Primers for gene one by one insertion, After having realized that instead of constructing a complex of wished genes and insert it to another does notlead to anything, We started to approach our goal by changing our mechanism and switching to one by one insertion of genes.

Started using the blue-white screening technique while transforming samples to the plates.

Lab technician day.Prepared LB media, different volume tips for autoclaving.

No colonies present of p8, p9, p10. Restared with digestion of those 3 for 3 days with ECORi PSTI restrictases.

p37, p39, p41. LIac mediated yeast transformation. First transformation into the yeast. Successful.

last transformation to the yeast.

Characterization of the biobrick BBa\_K2576009 (pRPL18B promoter).

Strains used:

* YST1 (CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 [pRS306, ApaI])
* YST2 (CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 [pRS306 pRPL18B-EGFP-tCYC1,ApaI])
* YST3 (CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 [pRS306 pTDH3-EGFP-tCYC1,ApaI])

[Read the whole notebook in our benchling](https://benchling.com/s/etr-gWRVXVpu9z2pgLl1GxAc)

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# Team:Tartu TUIT/Experiments

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* Project
  + [Description](/Team%3ATartu_TUIT/Description)
  + [Design](/Team%3ATartu_TUIT/Design)
  + [Modeling](/Team%3ATartu_TUIT/Model)
  + [Parts](/Team%3ATartu_TUIT/Parts)
  + [Results](/Team%3ATartu_TUIT/Results)
* Laboratory
  + [InterLab](/Team%3ATartu_TUIT/InterLab)
  + [Experiments](/Team%3ATartu_TUIT/Experiments)
  + [Notebook](/Team%3ATartu_TUIT/Notebook)
  + [Safety](/Team%3ATartu_TUIT/Safety)

* People
  + [Team](/Team%3ATartu_TUIT/Team)
  + [Attributions](/Team%3ATartu_TUIT/Attributions)
* Human Practices
  + [Human Practices](/Team%3ATartu_TUIT/Human_Practices)
  + [Public Engagement](/Team%3ATartu_TUIT/Public_Engagement)
  + [Collaborations](/Team%3ATartu_TUIT/Collaborations)

EXPERIMENTS

## Restriction Digest

20 minutes (Incubation time is not considered)

* Water
  to 50μL
* Buffer (NEB or FastDigest Green Buffer (10X))
  5 μL
* DNA Template
  up to 5 μL
* Restriction enzymes
  2.5 µL of each restriction enzyme
* FastAP
  1.25 µL

* Thermostate (37oC)
* Cold body (To keep restriction enzymes)

1. Keep all reagents on ice.
2. Make a reaction mix into 20 µL or 50 µL (depends on your restriction) volume in a microfuge tube.
3. Add reagents in following order: water, buffer (NEB or FastDigest Green Buffer (10X)), DNA template, restriction enzyme, FastAP (if recommended by your supervisor).
4. Mix gently and spin down.
5. Incubate at 37oC in a thermostate for 1 hour or overnight (overnight for NEB Buffer & at maximum 1 hour for FastDigest Green Buffer (10X)).
6. Analyze your results via gel electrophoresis.

1. <https://www.genscript.com/restriction-digestion-protocol.html>
2. <https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012556_FastDigest_ApaI_UG.pdf>

## PCR Purification

20 minutes

* FADF Buffer
* Wash Buffer
* Elution Buffer

* Heat block (55oC)
* Vortex
* FADF Column
* Collection Tube
* Centrifuge Machine

1. Excise the agarose gel with a clean scalpel.Remove the extra agarose gel to minimize the size of gel slice.
2. Transfer up to 300 mg of the gel slice into a microcentrifuge tube.The maximum volume of gel slice is 300 mg.
3. Add 500 µL of FADF Buffer to the sample and mix by vortexing.For > 2% agarose gel, add 1000 µL of FADF Buffer.
4. Incubate at 55oC for 5-10 minutes and vortex the tube every 2~3 minutes until the gel slice dissolved completely.During incubation, interval vortexing can accelerate the gel dissolved.Make sure that the gel slice has been completely dissolved before proceed the next step.After gel dissolved, make sure that the color of sample mixture is yellow. If the color is violet, add 10 µL of sodium acetate, 3M, pH 5.0. Mix well to make color of sample mixture turned to yellow.
5. Cool down the sample mixture to room temperature. And place FADF Column into a Collection tube.
6. Add 800 µL of sample mixture to the FADF Column. Centrifuge at 11,000 x g for 30 seconds, discard the flow-thorugh. If the sample mixture is more than 800 µL, repeat this step for rest of sample mixture.
7. Add 750 µL of Wash Buffer (ethanol added) to the FADF Column. Centrifuge at 11,000 x g for 30 seconds and discard the flow-through.Make sure that the ethanol (96 - 100%) has been added to Wash Buffer when first use.
8. Centrifuge again at full speed (18,000 x g) for an additional 3 minutes to dry the column matrix.Important step! The residual liquid should be removed thoroughly on this step.
9. Place the FADF Column to a new microcentrifuge tube.
10. Add 40 µL of Elution Buffer or ddH2O to the membrane center of the FADF Column. Stand the FADF Column for 1 min.
    Important step! For effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.Important: Do not elute the DNA using less than suggested volume (40 µL). It will lower final yield.
11. Centrifuge at full speed (~ 18,000 x g) for 1 min to elute DNA.

## DNA Concentration Measurement

5-10 miutes

* Water
* Sample
* Elution Buffer

* NanoDrop Microvolume Spectrophotometer

1. Choose Nucleic Acid from the menu
2. Open the sample arm. Clean the flat surfaces from both sides.
3. Pipette around 1~2 µL of water to the sample surface. Click Ok and machine will be ready to work.
4. Add around 1~2 µL of Elution Buffer to make Blank measurement. Click “Blank”. Clean the surfaces from both sides.
5. Pipette around 1~2 µL of sample to sample surface. Click “Measurement”. Now, sample result is ready.

## Agarose Gel preparation

1 hour

* TAE Buffer
* Agarose
* Ethidium Bromide

* Conical flask
* Gel caster
* Comb

1. Add 100 mL of TAE Buffer to conical flask
2. Weigh 2 grams of agarose and add to 100 mL of buffer solution (2% agarose gel)
3. Keep in oven until agarose is completely melted
4. Take the solution from oven
5. Add ethidium bromide (4 µL of ethidium bromide per 100 mL of TAE Buffer)
6. Poor the solution to a gel cluster
7. Place the comb

1. <http://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=2>

## Bacterial Transformation

1 hour 30 minutes – 2 hours (Incubation time is not considered)

* Strain E.coli DH5a
* Plasmid DNA
* LB liquid medium
* Ice
* Selection plates

* hermostate 37oC
* Heat block (42oC)

1. Take the competent cells out of -80C fridge and put them on ice and let them melt
2. Take 1 µL of plasmid DNA and put it in a separately labelled eppendorf
3. Add 50 µL of the cells, resuspend gently
4. TLeave it on ice for 30 minutes
5. Add ethidium bromide (4 µL of ethidium bromide per 100 mL of TAE Buffer)
6. Poor the solution to a gel cluster
7. Heat shock at 42oC for 2 minutes
8. Put them on ice for 2 minutes
9. Add 400 µL of LB medium into the eppendorfs, put them all in a flask and shake at 37oC for 30 minutes
10. Take selection plates from cold room and leave them on the table (label them and add around 10 glass beads to each plate)
11. Take eppendorfs out and centrifuge them at 6000 rpm for 60 seconds
12. Remove supernatant and resuspend cells in the remaining liquid
13. Pipette cells onto the selection plates and shake them until surface is dry
14. Transfer the glass beads into the bid container containing ethanol
15. Let cells grow at 37oC overnight in thermostate

1. <http://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=2>

[Read the whole list of experiments in PDF format](https://static.igem.org/mediawiki/2018/f/f7/T--Tartu_TUIT--protocols.pdf)

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# Team:Tartu TUIT/Results

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* Project
  + [Description](/Team%3ATartu_TUIT/Description)
  + [Design](/Team%3ATartu_TUIT/Design)
  + [Modeling](/Team%3ATartu_TUIT/Model)
  + [Parts](/Team%3ATartu_TUIT/Parts)
  + [Results](/Team%3ATartu_TUIT/Results)
* Laboratory
  + [InterLab](/Team%3ATartu_TUIT/InterLab)
  + [Experiments](/Team%3ATartu_TUIT/Experiments)
  + [Notebook](/Team%3ATartu_TUIT/Notebook)
  + [Safety](/Team%3ATartu_TUIT/Safety)

* People
  + [Team](/Team%3ATartu_TUIT/Team)
  + [Attributions](/Team%3ATartu_TUIT/Attributions)
* Human Practices
  + [Human Practices](/Team%3ATartu_TUIT/Human_Practices)
  + [Public Engagement](/Team%3ATartu_TUIT/Public_Engagement)
  + [Collaborations](/Team%3ATartu_TUIT/Collaborations)

RESULTS

## Introduction

In order to determine the strength of pRPL18B promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. We have chosen pTDH3 as a positive control due to its wide use in yeast research. The empty plasmid was used as a negative control.

## MATERIAL AND METHOD

Plasmids used to conduct the experiment are listed in table 1.

Table 1. Plasmids created

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | | Insert | | |  |
| number | Plasmid name | Promoter | Gene | Terminator | backbone |
| 1 | pRS306 pTDH3-EGFP-tCYC1 | TDH3 | EGFP | tCYC1 | pRS306 || 2 | pRS306 pRPL18B-EGFP-tCYC1 | pRPL18B | EGFP | tCYC1 | pRS306 |
| 3 | pRS306 | - | - | - | pRS306 |

After construction of plasmids was finished, we have transformed S. cerevisiae CEN.PK-2-1C strain with plasmids listed above to create the strains (table 2).

Table 2. S. cerevisiae strains created.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Strain name | Genotype | Plasmid integrated |
| positive control | YST1 | CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2 | pRS306 pTDH3-EGFP-tCYC1 |
| Negative control | YST2 | CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2 | pRS306 |
 Test | YST3 | CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2 | pRS306 pRPL18B-EGFP-tCYC1 |

We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
All strains were pregrown overnight at 30°C in liquid –URA/2%Glc media, resuspended in fresh -URA/2%Glc media to OD600 ca. 1 and distributed to 96 well plate (clear flat bottom). 3 replicates from each strain were used. 100 µL of cells were added to each well. As a reference to OD600, fresh -URA/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.

Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | pRPL18B | | | | | |  |
|  |  | Negative control | Positive control | Colony 1 | Colony 2 | Colony 3 | Colony 4 | Colony 5 | Colony 6 | -Ura/2% Glc |
|  |  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Replicate 1 | A |  |  |  |  |  |  |  |  |  |
| Replicate 2 | B |  |  |  |  |  |  |  |  |  |
| Replicate 3 | C |  |  |  |  |  |  |  |  |  |

As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according with 2018 iGEM InterLab Study [2]. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements. The layout of the 96 well plates is given in table 4.

Table 4. The layout of the plate for Fluorescein standard curve measurements.

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Fluorescein µM |  | 0,313 | 0,157 | 0,078 | 0,039 | 0,0196 | 0,0098 | 0,0049 | 0,0024 | 0 |
|  |  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Replicate 1 | A | 79055 | 39438 | 20511 | 10034 | 5020 | 2731 | 1302 | 745 | 78 |
| Replicate 2 | B | 80361 | 40466 | 20735 | 10572 | 5367 | 2751 | 1371 | 784 | 86 |
| Replicate 3 | C | 76390 | 38680 | 19692 | 10112 | 5078 | 2719 | 1462 | 866 | 111 |
| Replicate 4 | D | 77470 | 38863 | 19833 | 10170 | 5235 | 2773 | 1344 | 765 | 121 |

## Results

The results are presented in figure 1. As it turned out, the pRPL18B promoter is weaker than pTDH3 but still active. This corresponds to the data from the source article [[1]](#ref-1)

Figure 1

![](https://static.igem.org/mediawiki/2018/a/a5/T--Tartu_TUIT--results1.svg)

*Figure 1.1.*

## CONCLUSION

We think the characterisation of the promoters is necessary to provide researchers with useful information and toolbox of promoters of different strength to better optimise synthetic pathways.

1. Denby, C. M., Li, R. A., Vu, V. T., Costello, Z., Lin, W., Chan, L. J. G., ... & Scheller, H. V. (2018). Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer. Nature communications, 9(1), 965.
2. https://static.igem.org/mediawiki/2018/0/09/2018\_InterLab\_Plate\_Reader\_Protocol.pdf, calibration 3

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