iGEM 2022 — Team sorbonne-u-paris

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# iGEM SORBONNE

NAWI

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Notebook

Our laboratory notebook

# Access to dates

[July 11th](#1)
[July 12th](#2)
[July 19th](#3)
[July 20th](#4)
[July 21h](#5)
[July 22th](#6)
[July 25th](#7)
[July 26th](#8)
[July 27th](#9)
[August 5th](#10)
[August 8th](#11)
[August 9th](#12)
[August 22th](#13)
[August 23th](#14)
[September 9th](#15)
[September 21th](#16)
[September 30th](#17)
[October 3rd](#18)
[October 7th](#19)
[October 11th](#20)

# Monday July 11th 2022

TAP and TAP agar media preparation (Tris-minimal medium) :

* Tap medium for 1 L (adjust final pH to 7.0 with HCl) :
  1M Tris base (e.g. Trizma) 20mL
  Phosphate buffer II  1.0mL
  Solution A  10.0mL
  Hutner’s trace elements 1.0mL
  Glacial acetic acid  1.0mL
  ‍
* Phosphate buffer II (for 100mL) :
  K2HPO4  10,8g
  KH2PO4 5,6g
  ‍
* Solution A (for 500mL) :
  NH4Cl  20g
  MgSO4 7H20 5g
  CaCl2 2H2O  2,5g
  ‍
* For TAP-agar : 15g/L agar

PCR on p0-260 in order to get pPSAD in A2-B1 position :

PCR on p0-260 pPSAD + 5’UTR with the GG147 / GG191 primers in order to get an insert of the pPSAD promoter in A2-B1 :

* GG147 : ttgaagacaactcatgacCACACACCTGCCCGTCTGCC
  (PPSAD+5'UTR\_PSAD\_A2\_Fw)
  ‍
* GG191 : TTGAAGACAACTCGATGGGGCTTGTTGTGAGTAGCAGTGGGG
  (PPSAD+5'UTR\_PSAD\_B1\_Rv)
  ‍

The concentration of p0-260 was 100,4ng/µL and the PCR product expected to get 850bp.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture.png)![](https://static.igem.wiki/teams/4316/wiki/notebook/capture1.png)

# Tuesday July 12th 2022

PCR on p0-260 in order to get pPSAD in A2-B1 position :

From a solution of p0-260 at 100,4 ng/µL, we made a dilution in order to have a concentration at 2 ng/µL
→ Dilution of p0-260 : 40 µL of water + 0,8 µL of sample.
The PCR product is expected to get 850bp.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture2.png)

This time, we also did a negative control with 1 µL of water instead of 1 uL of template DNA

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture3.png)

PCR on THB1 in order to have THB1 in B3-B5 position :

The concentration of THB1 was 51,7 ng/µL and the PCR product expected to get 458 bp.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture4.png)

We also did a negative control with 4 µL of water instead of 4 uL of template DNA.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture5.png)

Both PCRs didn’t work, probably because of an experimental handling error.

# Tuesday July 19th 2022

PCR on p0-260 in order to get pPSAD in A2-B1 position

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture6.png)

We also did a negative control with 4 µL of water instead of 4 µL of template DNA.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture7.png)

The band obtained is lower than expected. We have to redo the experiment.

PCR on THB1 in order to have THB1 in B3-B5 position

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture8.png)

We also did a negative control with 6 µL of water instead of 6 µL of template DNA.

# Wednesday July 20th 2022

Because we couldn’t succeed to do a proper PCR on p0-260, we decided to change the plasmid and use p001 instead.

PCR on p001 in order to get pPSAD in A2-B1 position :

From a solution of p0-001 at 224 ng/µL, we made a dilution in order to have a concentration at 2 ng/µL.
→ Dilution of p0-001 : 50 µL of water + 0,45 µL of sample.
The PCR product is expected to get 850bp.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture9.png)

We also did a negative control with 4 µL of water instead of 4 µL of template DNA.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture10.png)

Result of the PCR on THB1

This morning, we verified the PCR result on THB1 that we did yesterday with an electrophoresis.

Extraction, Purification and Dosage :
We wanted to extract 30 ng/uL of THB1 from the agarose gel
But after the purification, we only got 1,3 ng/uL
→ we need to do it again by changing some parameters

PCR on THB1 in order to have THB1 in B3-B5 position

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture11.png)![](https://static.igem.wiki/teams/4316/wiki/notebook/capture12.png)

# Thursday July 21th 2022

Extraction, Purification and Dosage for the PCR on P001.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture13.png)

The blank was made with Buffer NE

# Friday July 22th 2022

MoClo of pPSAD A2-B1 with pAGM9121 :

The software gives the volume to use based on the concentration measured on the Nanodrop.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture14.png)

The volumes of those two tubes are 14 µL. Plus the ligase mix of 6 µL, the final volume is 20 µL.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture15.png)

Then, we do a PCR with these parameters, without the final step (20 min at 65°).

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture16.png)

Transformation of E.coli with p0-pPSAD :

We take 25 µL of competent E.Coli (stored at -80°) and add 4 µL of ligation product (p0-pPSAD) :
15 minutes on ice / 45 secondes at 42°C / 10 min at 4°C.
We add 500 µL of LB breth.
centrifugation : 600 rpm at 37°C for 1 hourthrow away the supernatant, then centrifuge at 400 g for 2 minutes.
Spread out on a petri dish of LB Xgal+Ampicilline (100-200µL) : Incubate overnight at 37°C.
‍
The bacteria with the plasmid containing the insert appears white.
‍

# Monday July 25th 2022

For this gel, we used TAE instead of TBE and the electrophoresis chamber already contained some TBE so the gel didn’t work.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture18.png)

Transformation of E.coli with p0-pPSAD :

3 mL LB
3 µL antibiotic Spec (1000X → 1X)
In each tube, we put one white colony of E.coli. Then, the tubes are placed in the agitator at 37°C overnight.

PCR on THB1 in order to have THB1 in B3-B5 position :

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture19.png)

We also did a negative control with 4 µL of water instead of 4 µL of template DNA.

The PCR machine failed during the first step but we decided to continue the experiment anyway. Unfortunately, the gel didn’t work.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture20.png)

# Tuesday July 26th 2022

Mini prep of p0pSAD :

Purification :
‍

* Centrifugation of the E.coli colonies (4000 g for 3 min)
* Throw away the supernatant in a tube “LB waste”
* For the cellular lysis : Add 250 µL of buffer A1 (in the fridge, contains the RNase) to resuspend and then put the mix into a 1,5mL Eppendorf tube. Add 250 µL of buffer A2, mix the tube by reversing it, wait 5 min at room temperature. Add 300 µL of buffer A3, mix the tube by reversing it until the blue color is gone.
* Centrifugation of the lysate (11000 g for 5 min)
* Bind DNA : Place the column into a collection tube of 2mL. Put 700 µL (maximum of capacity) of supernatant in the column. Centrifugation at 11000 g for 1 min. Throw away the content of the collection tube into a tube noted “Chemical waste”. If there is more than 700 µL, repeat these steps with the same column.
* Washing : Add 500 µL of buffer AW (**this buffer needs to be heated at 50°C!**). Centrifugation at 11000 g for 1 min. Add 600 µL of buffer A4. Centrifugation at 11000 g for 1 min. Throw away the content of the collector tube into the “Chemical waste” tube.
* Drying of the column : Centrifugation at 11000 g for 2 min. Throw away the collector tube after emptying it.
* Elution : Place the column into a 1,5mL Eppendorf tube. Add 50 µL of elution buffer and wait 1 min at room temperature. Centrifugation at 11000 g for 1 min

Digestion :
‍
We measured the concentration of our tubes with the Nanodrop. We did a blank of 1,5 µL elution buffer. All our tubes have a concentration around 30 ng/µL so they are usable for digestion.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture21.png)

PCR on THB1 in order to have THB1 in B3-B5 position :

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture22.png)

We also did a negative control with 8 µL of water instead of 8 µL of template DNA.

# Wednesday July 27th

Digestion :

Same process as before but this time we used the BsaI enzyme and did a control with the plasmid PAGM9121 (in the 2nd slot).
‍

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture23.png)

PCR on THB1 in order to have THB1 in B3-B5 position :

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture24.png)

We used 3 times the quantities listed above in the table. We sorted these quantities in 3 tubes : one containing 50 µL and two containing 25 µL. We’ve also made a control without DNA. We added 10 µL of 6X loading purple in the 50 µL PCR mix tube and 5 µL in the 25 µL PCR mix tube.

In the following blot :
Well 1 :O'GeneRuler 1 kb Ladder (4 µL)
Well 2 (takes up the equivalent of two spaces/wells): control = no DNA (20 µL)
Well 3 (takes up the equivalent of two spaces/wells): 60 µL of the PCR mix
Well 4 : 20 µL of the PCR mix

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture25.png)

# Friday August 5th

PCR on p0-006 in order to get tPSAD in B6 position :

From a solution of p0-006 at 131 ng/µL, we made a dilution in order to have a concentration at 4 ng/µL.
The PCR product is expected to get 584 bp.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture26.png)

We also did a negative control with 5 µL of water instead of 5 µL of template DNA.

# Monday August 8th

Migration of p0-006 PCR product on agarose gel and purification

Purification  of tPSAD :  29 ng/µL (ratio of 0,69).
It may have interferences because there was ten times the normal amount of  BET in the gel, and the final volumes of PCR product between the control and the sample were different.

We also did a negative control with 5 µL of water instead of 5 µL of template DNA.

# Tuesday August 9th

MoClo reaction between the PCR product of tPSAD and the backbone pAGM9121

MoClo level 0. The objective is to get tPSAD in B6 position in order to make our test construction with the insulator sequence.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture27.png)

Order of THB1 and Atgypsy in A1 and C1 on Twist Bioscience

Indeed, we were not able to do PCR on genomic DNA with such primers (with a long floating tail, because of the restriction and fusion sites), so we had to order them on Twist Bioscience.

PCR on p0-35 in order to get mVenus in B2-B5 position

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture29.png)![](https://static.igem.wiki/teams/4316/wiki/notebook/capture30.png)

PCR product should be around 800 bp.

PCR de vérification des constructions de niveau 0 mVenus et tPSAD

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture31.png)

In order from left to right: mVenus(B2-B5) 1 to 3 and tPSAD(B6) 1 to 3 digested by BsaI; and finally the 1kb+ ladder.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture32.png)

Expected size for mVenus: 815 bp.
Expected size for tPSAD: 564 bp.

# Monday August 22th

Liquid culture of chlamydomonas reinhardtii ( V = 50 mL) for culture experiments. All cells were cultured in light-exposed conditions, under Xg of agitation and at 20°C.

# Tuesday August 23th

We received THB1 aligned primers (forward and reverse).
We added :
- 513 uL of water for THB1\_align\_FW
- 466 uL of water for THB1\_align\_RV
From this solution of primers at 100 ug/L, we made a dilution in order to have a concentration at 10 ug/L.
→ 90 µL of water + 10 µL of sample

PCR on THB1 in order to have THB1 in B3-B5 position

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture33.png)

We also did a negative control with 8 µL of water instead of 8 µL of template DNA.

# Friday September 9th

Inoculation of UVM4 strain of *Chlamydomonas reinhardtii* from solid TAP-agar to 100mL of liquid TAP. All cells were cultured in light-exposed conditions, under Xg of agitation and at 20°C.

# Friday September 21th

Inoculation of 2 cultures of UVM4 strain of *Chlamydomonas reinhardtii* from 1 mL and 0.5 mL of September 9th’s culture. All cells were cultured in light-exposed conditions, under Xg of agitation and at 20°C.

# Friday September 30th

Inoculation of 100mL of TAP media with 1/100th and 1/10th dilutions of September 9th’s cultures. All cells were cultured in light-exposed conditions, under Xg of agitation and at 20°C.

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture34.png)

Standard TAP media composition

100mL of 7 TAP media were prepared, deviating from the standard recipe in the following ways :
- 1 did not differ from the recipe
- 1 had no acetate in the recipe
- 1 halved the normal amount of acetate in the recipe (9.15 µM)
- 1 doubled the normal amount of acetate in the recipe (36.6 µM)
- 1 with 0.1 µM of iron added to the final iron content (0.18 µM + 0.1 µM = 0.28 µM)
- 1 with 0.25 µM of iron added to the final iron content (0.43µM)
- 1 with 20 µM of iron added to the final iron content (20.18 µM)

# Monday October 3rd

Cell count was determined from 30/09’s cultures using a X cell-counter reading red fluorescence from XXX :
- d10 numbered 1.59\*10^7 cells/mL
- d100 numbered 6.13\*10^6 cells/mL

Triplicates of 30mL of each TAP media prepared on September 30th were inoculated with 8.16 µL of the d100 culture, as to have an initial concentration of 0.5\*10^5 cells/mL.

Each triplicate was then installed in a bioreactor for culture over a week in different conditions, that can be recapped in the following table :

![](https://static.igem.wiki/teams/4316/wiki/notebook/capture35.png)

All cells were cultured under Xg of agitation and at 20°C.
Day corresponds to light-exposed growth conditions and night to dark growth conditions

# Friday October 7th

Partial growth curve data downloaded from bioreactor and transmitted to the modeling team. Culture to continue over the weekend under the same conditions.

# Tuesday October 11th

End of cell culture after ≈ 192 hours and transmission of photobioreactor measurement data to modelling team.

![](https://static.igem.wiki/teams/4316/wiki/notebook/lcqb.png)

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### About Us

We are the 2022 team of Sorbonne University iGem. With an interdisciplinary team of students we aim to improve the world with our project NAWI !

### Follow Us

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### Contact Us

Sorbonne\_U\_Paris
igem.sorbonne.2022@gmail.com

© 2022 - Content on this site is licensed under a

[Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at

[gitlab.igem.org/2022/sorbonne-u-paris](https://gitlab.igem.org/2022/sorbonne-u-paris)

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# iGEM SORBONNE

NAWI

Parts

Parts

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Awards

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Engineering

Synthetic biology is built around the principles of engineering, in particular the design, build, test and learn cycle

# Design

On the biological side, the idea was to increase expression of a THB1 (truncated hemoglobin 1) gene in cells. Indeed, heme-associated iron is the best absorbed in the intestine. Therefore, the controlled overexpression of a heme protein capable of binding iron was the challenge of our project.
‍
We decided to express the THB1 transgene in a Nit2- strain (UVM4). The THB1 protein is regulated by the transcription factor Nit2, which is the major transcription factor in nitrate assimilation. By using a Nit2- strain, THB1 expression naturally present in *C.reinhardtii* is prevented. We can therefore have a completely controlled expression of the transgene.

In order to maximize this control, the use of insulator sequences was chosen. Insulator sequences are found in certain eukaryotes, and make it possible to protect a gene or a group of genes from the effects produced by neighboring regulatory sequences. During the stable transformation, endogenous transcriptional enhancers can interact with promoters in artificial systems, or genomic heterochromatin can spread, which leads to an obstacle in the expression. In order to obtain a stable expression, the use of insulator sequences framing our gene system is therefore essential.

![](https://static.igem.wiki/teams/4316/wiki/engineering/sch-ma-insulators.gif)

However, the existence of insulator sequences in *C.reinhardtii* has never been demonstrated. Several solutions were therefore available to us.

First of all, the *gypsy* insulator, which is the most characterized enhancer-blocking insulator. It is found within the 5’-untranslated region of the *gypsy* retrotransposon from *Drosophila* (∼350bp). This insulator sequence works with zinc finger proteins Su(Hw) (Suppressor of Hairy-Wing), which recruit additional factors in order to form the “insulator body”. The *gypsy* insulator and other non-plant insulators have been shown to be functional in plants, such as transgenic *Arabidopsis thaliana*. This suggests the conservation of components involved in insulator activity in plants (ex: proteins). As *C.reinhardtii* is one of the greatest models for superior plant, insulator sequences such as the *gypsy* one could work in this organism.

The second solution was to find insulator sequences directly present in plants as *C.reinhardtii* is an eukaryotic photosynthetic organism. One of the plant insulator candidates is the *gypsy*-like sequence (2258bp) found in the *Arabidopsis thaliana* genome.  *‍*Finally, to complete the control of expression, we chose to use an inducible promoter to express THB1. Indeed, we want to grow our algae in bioreactors, and in order to get rid of the burden that our transgene might impose on *C.reinhardtii*'s metabolism that could impact its growth, we wanted to be able to choose when to induce the expression. Moreover, we wanted to grow our algae in the dark, so that there would be less chlorophyll, which has an impact on the bad taste of algae. Therefore, we choose to use a light inducible promoter, such as the high light-inducible *Dunaliella* LIP promoter  which is easy to use and does not require any addition to the medium.

Our final plasmid should therefore be composed of an origin of replication, the insulator sequences, the THB1 gene and an antibiotic resistance gene in order to facilitate the selection.

# Build

In order to build our plasmids we relied on the MoClo kit for *C.reinhardtii*. MoClo (Modular Cloning) is a standardized method based on the use of building blocks and Golden Gate cloning. Each standardized gene part (level 0) is designed and cloned for a specific position in the transcription unit (level 1). Several transcription units can be combined (level M) to allow the expression of several genes at the same time. The MoClo kit is therefore perfectly scalable, with easy exchange of parts, if designed for the same position, by adding specific sites.

When we first tried to construct the plasmid, we wanted to use the *Atgypsy* sequence as the insulator sequence. However, this sequence is very long (2258bp) and isolating it from the *Arabidopsis thaliana* genome or producing it via our sponsors was not easy. We therefore decided to express the *gypsy* sequence as well as its Su(Hw) gene sequence in our construction. Each standardized gene part is therefore manufactured or being manufactured to obtain our final plasmid.

# Test

Before testing our complete design, we first had to test and prove that the *gypsy* insulator sequence together with its Su(Hw) protein fulfilled its function in the model organism *C.reinhardtii*.
Two plasmids have been designed and are under construction. These are a test plasmid (pM-insulator) and a control plasmid (pM-control). The test plasmid consists of the insulator sequences, a gene encoding a fluorescence protein (mVenus) under the control of the constitutive promoter (pPSAD), and the gene encoding the Su(Hw) protein. While the control plasmid has only the fluorescence protein gene under the control of the constitutive promoter.
After transformation of the UVM4(Nit2-) strains with the two plasmids and culture in multi-well plates, the test is performed using a plate reader. The objective is to measure the fluorescence intensity of mVenus in colonies with the test plasmid and those with the control plasmid.
‍
The proof of concept is that the fluorescence intensity varies for colonies with the control plasmid and is constant for colonies with the test plasmid. Indeed, as the *gypsy* sequences isolate the transgene from the genomic context, the expression of the fluorescent protein should not vary.

![](https://static.igem.wiki/teams/4316/wiki/engineering/sch-ma-cycle-001.png)![](https://static.igem.wiki/teams/4316/wiki/engineering/sch-ma-cycle-002.png)

# Anticipated re-design

By furthering our research and discussing with local actors, we were able to anticipate possible redesigns of our system.

Our modelisation team works to provide to the iGEM community a growth model of Chlamydomonas reinhardtii.
Our objective was to model the growth of the wild type C. reinhardtii (uvm4 strain), in order to be able to compare this growth to that of our GMO once its construction is finished. Indeed, the idea is to see if the addition of genes to the genome does not cause a burden to the metabolism, which could have an impact on growth and therefore production.
The design is based on the growth of microalgae in different TAP media, the usual one, but also in some with different acetate or iron concentrations (cf. protocole, bioreactors culture). The purpose is to find the best media to grow our GMOs in order to get the highest production at the end.
To obtain the results we grew C. reinhardtii in Algenuity bioreactors in different conditions of light and media. After a week of culture, we were able to analyze our results and to implement our model. (see [our model](https://2022.igem.wiki/sorbonne-u-paris/model)
)

# Sources

1. (Kurbidaeva, Amina, and Michael Purugganan. “Insulators in Plants: Progress and Open Questions.” Genes vol. 12,9 1422. 16 Sep. 2021, doi:10.3390/genes12091422).
2. *(A gypsy-like sequence from Arabidopsis thaliana exhibits enhancer-blocking activity in transgenic plants Stacy D. Singer & Kerik D. Cox).*
3. (Park S, Lee Y, Lee JH, Jin E. Expression of the high light-inducible Dunaliella LIP promoter in Chlamydomonas reinhardtii. Planta. 2013 Dec;238(6):1147-56. doi: 10.1007/s00425-013-1955-4. Epub 2013 Sep 17. PMID: 24043576)
4. (Crook N, Abatemarco J, Sun J, Wagner JM, Schmitz A, Alper HS. In vivo continuous evolution of genes and pathways in yeast. Nat Commun. 2016;7: 13051. doi:10.1038/ncomms13051).

![](https://static.igem.wiki/teams/4316/wiki/engineering/nawi2-color-edited.png)

[Design](#design)
[Build](#build)
[Test](#test)

![](https://static.igem.wiki/teams/4316/wiki/engineering/lcqb-logo.png)

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### About Us

We are the 2022 team of Sorbonne University iGem. With an interdisciplinary team of students we aim to improve the world with our project NAWI !

### Follow Us

[![](https://static.igem.wiki/teams/4316/wiki/model2/logo-insta.jpg)](https://www.instagram.com/igemsorbonne/)
[![](https://static.igem.wiki/teams/4316/wiki/model2/logo-linkedin.png)](https://www.linkedin.com/company/igem-sorbonne/)

### Contact Us

Sorbonne\_U\_Paris
igem.sorbonne.2022@gmail.com

© 2022 - Content on this site is licensed under a

[Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at

[gitlab.igem.org/2022/sorbonne-u-paris](https://gitlab.igem.org/2022/sorbonne-u-paris)

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# iGEM SORBONNE

NAWI

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Experiments

Our lab work

You will find here the entries of the protocols used as well as those that we would like to carry out to continue our project.

* [Bioreactor Culture](https://static.igem.wiki/teams/4316/wiki/experiments/bio.pdf)
* [Buffers](https://static.igem.wiki/teams/4316/wiki/experiments/buff.pdf)
* [Chlamydomonas reinhardtii transformation](https://static.igem.wiki/teams/4316/wiki/experiments/chlam.pdf)
* [Functionning of insulators sequences](https://static.igem.wiki/teams/4316/wiki/experiments/func.pdf)
* [MoClo for Chlamydomonas reinhardtii](https://static.igem.wiki/teams/4316/wiki/experiments/moclo.pdf)
* [PCR](https://static.igem.wiki/teams/4316/wiki/experiments/pcr.pdf)
* [THB1 expression and iron concentration](https://static.igem.wiki/teams/4316/wiki/experiments/thb1.pdf)

![](https://static.igem.wiki/teams/4316/wiki/engineering/lcqb-logo.png)

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![](https://static.igem.wiki/teams/4316/wiki/engineering/logo-association-su-color.png)

### About Us

We are the 2022 team of Sorbonne University iGem. With an interdisciplinary team of students we aim to improve the world with our project NAWI !

### Follow Us

[![](https://static.igem.wiki/teams/4316/wiki/model2/logo-insta.jpg)](https://www.instagram.com/igemsorbonne/)
[![](https://static.igem.wiki/teams/4316/wiki/model2/logo-linkedin.png)](https://www.linkedin.com/company/igem-sorbonne/)

### Contact Us

Sorbonne\_U\_Paris
igem.sorbonne.2022@gmail.com

© 2022 - Content on this site is licensed under a

[Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at

[gitlab.igem.org/2022/sorbonne-u-paris](https://gitlab.igem.org/2022/sorbonne-u-paris)

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# iGEM SORBONNE

NAWI

Parts

Parts

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[Human Practices

Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)

Awards

Awards

[Education](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D)
[Entrepreuneurship](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27entrepreneurship%27%29%20%7D%7D)
[Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
[Plant](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27plant%27%29%20%7D%7D)
[Sustainable](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)
[Prizes](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27prizes%27%29%20%7D%7D)

Results

Our progress

# Wet Lab Results

Through our NAWI project we wanted to create a new innovative food source to feed humanity. To achieve this goal, we designed a plasmid to genetically modify the genome of the green algae *C. reinhardtii*. This plasmid is composed of a replication origin, *gypsy* insulator sequences, an inducible promoter, the THB1 gene, the Su(Hw) gene which codes for a protein working with *gypsy* and a hygromycin resistance gene. All these parts can be standardized for the MoClo kit for *C. reinhardtii*. The MoClo (Modular Cloning) kit allows an easy and modular building of plasmids.

![](https://static.igem.wiki/teams/4316/wiki/results/capture-removebg-preview-2.png)

We therefore started to standardize all these parts by adding fusion sites at their terminals by following the MoClo kit model (for level 0).
‍

![](https://static.igem.wiki/teams/4316/wiki/results/capture-removebg-preview-3.png)

Crozet and *al.*, 2018, A MoClo Toolkit Enabling Synthetic Biology in the Microalga *Chlamydomonas reinhardtii*

We started with the creation of the PSAD promoter part in the A2-B1 position. For this we did a PCR on a level 0 plasmid from the lab with pPSAD in A1-B1 position, thanks to floating tail primers.

**Example with mVenus in B2-B5 position**

We started by performing a PCR on p0-35, a level 0 plasmid with mVenus in B2 from the lab, in order to get it in B2-B5 to suit our design, thanks to floating tail primers (cf. [protocol PCR](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D))

![](https://static.igem.wiki/teams/4316/wiki/results/capture36.png)

*Agarose gel result for PCR from p0-35.

‍*As expected we obtained a 835nt PCR product, which was then extracted and purified using a kit from NEBⓇ.

This purified insert was used in a MoClo reaction in order to get mVenus in B2-B5 in a level 0 plasmid (p0-mVenus).In order to check if the MoClo reaction worked, we digested the obtained product with BsaI and we then made an agarose gel to see the results.

![](https://static.igem.wiki/teams/4316/wiki/results/capture37.png)

*Agarose gel result from the digestion of the product of the MoClo reaction with BsaI.

As expected we obtained a 815nt product, which corresponds to mVenus, the insert. And a product of 2057nt, which corresponds to the universal plasmid, the backbone.*

During our experimental period, we also had the time to make level 0 plasmids with pPSAD in A2-B1 position, and tPSAD in B6.
Unfortunately , we were unable to receive the THB1 gene sequence, and the gypsy insulator sequence from our sponsors. So we were not able to finish our build step…

![](https://static.igem.wiki/teams/4316/wiki/results/lcqb-logo.png)

![](https://static.igem.wiki/teams/4316/wiki/results/logo-cvec.png)

![](https://static.igem.wiki/teams/4316/wiki/results/logo-sorbonne-universit.png)

![](https://static.igem.wiki/teams/4316/wiki/results/logo-association-su-color.png)

### About Us

We are the 2022 team of Sorbonne University iGem. With an interdisciplinary team of students we aim to improve the world with our project NAWI !

### Follow Us

[![](https://static.igem.wiki/teams/4316/wiki/results/logo-insta.jpg)](https://www.instagram.com/igemsorbonne/)
[![](https://static.igem.wiki/teams/4316/wiki/results/logo-linkedin.png)](https://www.linkedin.com/company/igem-sorbonne/mycompany/)

### Contact Us

Sorbonne\_U\_Paris
igem.sorbonne.2022@gmail.com

© 2022 - Content on this site is licensed under a

[Creative Commons Attribution 4.0 International license.](https://creativecommons.org/licenses/by/4.0/)

The repository used to create this website is available at

[gitlab.igem.org/2022/sorbonne-u-paris](https://gitlab.igem.org/2022/sorbonne-u-paris)