iGEM 2021 — Team SCU-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SCU-China/Notebook

SCU-China

* [HOME](https://2021.igem.org/Team%3ASCU-China)
* PROJECT
  + [Description](https://2021.igem.org/Team%3ASCU-China/Description)
  + [Design](https://2021.igem.org/Team%3ASCU-China/Design)
  + [Results](https://2021.igem.org/Team%3ASCU-China/Results)
  + [Parts](https://2021.igem.org/Team%3ASCU-China/Parts)
* MODELING
  + [Modeling Overview](https://2021.igem.org/Team%3ASCU-China/Model)
  + [Protein Structure](https://2021.igem.org/Team%3ASCU-China/Protein_Structure)
  + [mRNA Stability](https://2021.igem.org/Team%3ASCU-China/mRNA_Stability_Modeling)
  + [Cas-based iFFL](https://2021.igem.org/Team%3ASCU-China/Cas-based_iFFL)
  + [Enzyme Kinetic](https://2021.igem.org/Team%3ASCU-China/Enzyme_Kinetic)
* HUMAN PRACTICE
  + [Integrated](https://2021.igem.org/Team%3ASCU-China/Human_Practices)
  + [Questionnaire](https://2021.igem.org/Team%3ASCU-China/Questionnaire)
  + [Communication](https://2021.igem.org/Team%3ASCU-China/Communication)
* LAB
  + [Safety](https://2021.igem.org/Team%3ASCU-China/Safety)
  + [Notebook](https://2021.igem.org/Team%3ASCU-China/Notebook)
  + [Protocols](https://2021.igem.org/Team%3ASCU-China/Protocol)
* TEAM
  + [Team Members](https://2021.igem.org/Team%3ASCU-China/Team_Members)
  + [Attributions](https://2021.igem.org/Team%3ASCU-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ASCU-China/Collaborations)
* MEDALS
  + [Contribution](https://2021.igem.org/Team%3ASCU-China/Contribution)
  + [Engineering](https://2021.igem.org/Team%3ASCU-China/Engineering)
  + [Implementation](https://2021.igem.org/Team%3ASCU-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3ASCU-China/Proof_Of_Concept)
  + [Measurement](https://2021.igem.org/Team%3ASCU-China/Measurement)
  + [Education](https://2021.igem.org/Team%3ASCU-China/Education)

# *Notebook*

#### Ackhowledgement

![](https://static.igem.org/mediawiki/2021/c/cd/T--SCU-China--sponsor.png)

#### Contact Information

* College of Life Science in Sichuan University,
  No.24 South Section
  1, Yihuan Road,
  Chengdu , China, 610065 | No.29 Jiuyanqiao Wangjiang Road, Chengdu,
  610064
* +86 13308049545
* scu\_igem@163.com

© 2021 SCU-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SCU-China/Engineering

SCU-China

* [HOME](https://2021.igem.org/Team%3ASCU-China)
* PROJECT
  + [Description](https://2021.igem.org/Team%3ASCU-China/Description)
  + [Design](https://2021.igem.org/Team%3ASCU-China/Design)
  + [Results](https://2021.igem.org/Team%3ASCU-China/Results)
  + [Parts](https://2021.igem.org/Team%3ASCU-China/Parts)
* MODELING
  + [Modeling Overview](https://2021.igem.org/Team%3ASCU-China/Model)
  + [Protein Structure](https://2021.igem.org/Team%3ASCU-China/Protein_Structure)
  + [mRNA Stability](https://2021.igem.org/Team%3ASCU-China/mRNA_Stability_Modeling)
  + [Cas-based iFFL](https://2021.igem.org/Team%3ASCU-China/Cas-based_iFFL)
  + [Enzyme Kinetic](https://2021.igem.org/Team%3ASCU-China/Enzyme_Kinetic)
* HUMAN PRACTICE
  + [Integrated](https://2021.igem.org/Team%3ASCU-China/Human_Practices)
  + [Questionnaire](https://2021.igem.org/Team%3ASCU-China/Questionnaire)
  + [Communication](https://2021.igem.org/Team%3ASCU-China/Communication)
* LAB
  + [Safety](https://2021.igem.org/Team%3ASCU-China/Safety)
  + [Notebook](https://2021.igem.org/Team%3ASCU-China/Notebook)
  + [Protocols](https://2021.igem.org/Team%3ASCU-China/Protocol)
* TEAM
  + [Team Members](https://2021.igem.org/Team%3ASCU-China/Team_Members)
  + [Attributions](https://2021.igem.org/Team%3ASCU-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ASCU-China/Collaborations)
* MEDALS
  + [Contribution](https://2021.igem.org/Team%3ASCU-China/Contribution)
  + [Engineering](https://2021.igem.org/Team%3ASCU-China/Engineering)
  + [Implementation](https://2021.igem.org/Team%3ASCU-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3ASCU-China/Proof_Of_Concept)
  + [Measurement](https://2021.igem.org/Team%3ASCU-China/Measurement)
  + [Education](https://2021.igem.org/Team%3ASCU-China/Education)

# Engineering success

## CRISPRa, Improvement of LacIAM in *V.n* and metabolic engineering

* [section-1](#section-1)
* [section-2](#section-2)
* [section-3](#section-3)

1. We achieved successful dCasphi-based CRISPRa in *Vibrio.natriegens*

We firslt apply
protein structure analysis to find the best activation domain for CRISPRa, the results showed that
**the α domain of RNAp from *Vibrio natriegens*** could accomplish the best gene
activation compared with others.

So we firstly fusion
V.nαdomain in the N domain of dCasΦ，but it impeded the gene expression while classic AD SoxS showed
a successful activation. We assumed that this may due to the fusion manner which was then proved by
both docking analysis and practical function tests that **Cas-linker-AD** is better
than our first design, AD-linker-Cas.

Moreover, by
comparing the modeling results from the I-TASSER server, we selected **a relatively more
appropriate linker** from the six linkers collected.

[Learn more](https://2021.igem.org/Team%3ASCU-China/Protein_Structure)

![](https://static.igem.org/mediawiki/2021/a/a4/T--SCU-China--scu-2021-ProteinModel-DBTL.png)

2. We decrease we leakiness of LacIAM-Ptac in Vibrio.natriegens by a RNA regulator

We observer that the
leakiness of LacIAM-Ptac in Vibrio.natriegens is extremely strong and nearly performs as a
constitutive promoter.

![](https://static.igem.org/mediawiki/2021/7/77/T--SCU-China--result-Figure1.1.8.2.png)

So we applied a RNA
regulator to arrest the translation of mSarlet-I so as to decrease the leakiness in the translation
level.

![](https://static.igem.org/mediawiki/2021/0/02/T--SCU-China--result-Figure1.1.8.3.jpg)

3. Our strain produced violacein and naringenin successfully

![](https://static.igem.org/mediawiki/2021/1/18/T--SCU-China--engineering3.png)

#### Ackhowledgement

![](https://static.igem.org/mediawiki/2021/c/cd/T--SCU-China--sponsor.png)

#### Contact Information

* College of Life Science in Sichuan University,
  No.24 South Section
  1, Yihuan Road,
  Chengdu , China, 610065 | No.29 Jiuyanqiao Wangjiang Road, Chengdu,
  610064
* +86 13308049545
* scu\_igem@163.com

© 2021 SCU-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SCU-China/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:SCU-China/Results

SCU-China

* [HOME](https://2021.igem.org/Team%3ASCU-China)
* PROJECT
  + [Description](https://2021.igem.org/Team%3ASCU-China/Description)
  + [Design](https://2021.igem.org/Team%3ASCU-China/Design)
  + [Results](https://2021.igem.org/Team%3ASCU-China/Results)
  + [Parts](https://2021.igem.org/Team%3ASCU-China/Parts)
* MODELING
  + [Modeling Overview](https://2021.igem.org/Team%3ASCU-China/Model)
  + [Protein Structure](https://2021.igem.org/Team%3ASCU-China/Protein_Structure)
  + [mRNA Stability](https://2021.igem.org/Team%3ASCU-China/mRNA_Stability_Modeling)
  + [Cas-based iFFL](https://2021.igem.org/Team%3ASCU-China/Cas-based_iFFL)
  + [Enzyme Kinetic](https://2021.igem.org/Team%3ASCU-China/Enzyme_Kinetic)
* HUMAN PRACTICE
  + [Integrated](https://2021.igem.org/Team%3ASCU-China/Human_Practices)
  + [Questionnaire](https://2021.igem.org/Team%3ASCU-China/Questionnaire)
  + [Communication](https://2021.igem.org/Team%3ASCU-China/Communication)
* LAB
  + [Safety](https://2021.igem.org/Team%3ASCU-China/Safety)
  + [Notebook](https://2021.igem.org/Team%3ASCU-China/Notebook)
  + [Protocols](https://2021.igem.org/Team%3ASCU-China/Protocol)
* TEAM
  + [Team Members](https://2021.igem.org/Team%3ASCU-China/Team_Members)
  + [Attributions](https://2021.igem.org/Team%3ASCU-China/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3ASCU-China/Collaborations)
* MEDALS
  + [Contribution](https://2021.igem.org/Team%3ASCU-China/Contribution)
  + [Engineering](https://2021.igem.org/Team%3ASCU-China/Engineering)
  + [Implementation](https://2021.igem.org/Team%3ASCU-China/Implementation)
  + [Proof of Concept](https://2021.igem.org/Team%3ASCU-China/Proof_Of_Concept)
  + [Measurement](https://2021.igem.org/Team%3ASCU-China/Measurement)
  + [Education](https://2021.igem.org/Team%3ASCU-China/Education)

# *Results*

* [section-1](#section-1)
* [section-2](#section-3)
* [section-3](#section-5)
* [section-4](#section-7)
* [section-5](#section-9)
* [section-6](#section-11)

..

1. Characterization of Different Regulator and Reporter

Different regulator
parts will influence the expression of genes in sharp dgree. For example, we got a database (N=2500)
from Ailurus, and the expression level between different combination of regulatory parts can differ
600 times.

![](https://static.igem.org/mediawiki/2021/c/c3/T--SCU-China--rf1.png)

..

2. Optimization of Protocols about Constructing Collection and Transformation

2.1 Process of constructing golden-gate-based
collection with low cost

We applied
TEDA to construct our collection. In the mixture of TEDA, only T5 exonuclease is added, so
it’s cheap for every lab and especially iGEM teams.

What’s more, sfGFP may not work in V.n as shown in Figure 1.2.1.1, so we use ccdB as the counter selection marker.

![](https://static.igem.org/mediawiki/2021/1/1d/T--SCU-China--result-Figure1.2.1.1.png)

Figure1.2.1.1

2.2 Improvement of natural transformation of V.n

![](https://static.igem.org/mediawiki/2021/0/06/T--SCU-China--result-Figure1.2.2.1.jpg)

Figure1.2.2.1

In Figure 1.2.2.1 , 150ug/ml streptomycin with tDNA shows “high efficiency”, but the sequencing result shows that they are faker! (Figure1.2.2.2)

![](https://static.igem.org/mediawiki/2021/4/4f/T--SCU-China--fi.png)

Figure1.2.2.2

..

3. Synthesis of naringenin and violacein in BL21 and Vibrio naringenin

Violacein and naringenin are classic natural products, and have been synthesized widely in different chassis organisms. We believe that the synthesis of these two products is a important step before a chassis organism could be accepted.

3.1 Synthesis of naringenin in Vibrio natriegens

Due to the lack knowledge of metabolic flux in Vibrio natriegens, we applied mathematic model and use parts from SSR collection to achieve naringenin synthesis in Vibrio natriegens, and the engineering is successful (as shown in Figure. 1.3.1.1, the first sample is 500μM positive control).

![](https://static.igem.org/mediawiki/2021/f/ff/T--SCU-China--3.1.png)

Figure. 1.3.1.1

3.2 Synthesis and analysis of violacein in BL21 and Vibrio natriegens

Then we try to synthesis violacein in BL21 and Vibrio natriegens.

The results are shown below.

![](https://static.igem.org/mediawiki/2021/d/d4/T--SCU-China--3.2.png)

Figure. 1.3.2.1

Interestingly, we found that their colony performance is vast different High copy number VioABCDE perform better in V.n, bu not BL21, this is to the contrary of production

![](https://static.igem.org/mediawiki/2021/6/6e/T--SCU-China--3.3.png)

Figure. 1.3.2.2

..

4. Engineering the CRISPRa Construction to Achieve the Best Efficiency

In prokaryote, most
of the CRISPRa strategies are based on the activation domains (AD) which could recruit components of
RNAp, so as to direct the work of holoenzyme. Until now, three manners of recruiting ADs nearby
promoter have been demonstrated successful gene activation as shown in Figure 1.4.

Most of related
CRISPRa research directly fused AD with dCas9 or dCas12a and usually achieve the best efficiency.
Another popular manner is applying RNA-binding protein and special RNA adapter such as MCP-MS2 and
γN22plus-BoxB. This manner could achieve the same activation level compared with direct fusion. In a
latest research, SYNZIP was used to recruit AD noncovalently and also achieve a high activation.

Here, we fused four
ADs and explored their capacity in the C-fusion or N-fusion manner with the integration of
modelling（[https://2021.igem.org/Team:SCU-China/Protein\_Structure](https://2021.igem.org/Team%3ASCU-China/Protein_Structure)）.
We found that α domain of V.n’s RNAp achieved the best gene activation.

![](https://static.igem.org/mediawiki/2021/c/ca/T--SCU-China--result-Fig3.1.png)

Figure 1.4

4.1 Significant difference between ADs and
fusion
manners

We chose four
activation domains to fuse in the N-domain of dCasΦ to test CRISPRa, and fusion protein was
expression by J23104-B0034, while crRNA was placed under aracPBAD promoter. 0.1%,0.5% and 1%
of Arabinose were applied to induce the transcription of crRNA.Unfortunately, only classic
SoxS showed a slight gene activation compared with other 3 combinations, as shown in
Figure 1.4.1.1. What’s more, the V.n α, which was estimated to perform CRISPRa best impeded the
expression of mSarlet-I

.
![](https://static.igem.org/mediawiki/2021/0/00/T--SCU-China--result-Figure3.1.1.png)

Figure 1.4.1.1

After
reference research, we assumed that the fusion manner, or fuse AD in the N-domain of dCasΦ
or in the C-domain of dCasΦ is important, and our model result backed up our suppose.

Then we build
plasmids for dCasΦ-linker-ADs, most of the constructions showed gene activation except P.a
α-NTD （Figure 3.1.2）. This is interesting too, because P.a α-NTD was declared better then
E.c α-NTD.

![](https://static.igem.org/mediawiki/2021/6/6d/T--SCU-China--result-Figure3.1.2.png)

Figure 1.4.1.2

4.2 Successful activation by three different
recruit manners

![](https://static.igem.org/mediawiki/2021/e/e3/T--SCU-China--rf322.png)

Figure 1.4.2.1

Then we test
the efficiency of CRISPRa in three different manner and two different linker, use 1%
L-arabinose to induce transcription of crRNA. We found that the best one is the
dCasΦ-3XGGGS-AD as our model suggested, so we applied dCasΦ-3XGGGS-AD to accomplish the
following test.

4.3 Different activation efficienct on
PAM-sites
upstream of TSS

Then we
applied dCasΦ-linker-V.n α to explore the concrete influence of distance between PAM site
and TSS (transcription start site). Two spacer sequence were chose as showed in blue
background as shown in Figure 3.3.2.1. To construct different plasmids with different
up-elements, we apply blunt ligation.

![](https://static.igem.org/mediawiki/2021/e/ea/T--SCU-China--rf3321.png)

Figure 1.4.3.1

The result is
shown in Figure 1.4.3.2

![](https://static.igem.org/mediawiki/2021/5/5c/T--SCU-China--rf3322.png
)

Figure 1.4.3.2

..

5. CRISPRi based Toggle Switch

5.1 Measurement of bistability

CRISPRi was
implemented by targeting the dCasφ-crRNA complex within the CDS of reporter genes. The
effector module blocks the RNA-polymerase and inhibits the transcription process. Our Toggle
switch was measured in E. coli DH5α for up to 40 hours over four phases. TS was successively
accessed with the addition of the first inducer (aTc), followed by no addition, the second
inducer (Ara), and finally no inducer again. Data was collected every ten hours by taking
120 μl of bacterial solution into 96-well plates then measured by the micro plate reader.
Each data point represents the mean of three biological replicates.

The results
showed that there was no states switch in the expression intensity of sfGFP. And mSarlet-I
expression intensity only showed a steady state in the ‘LOW’ state.

![](https://static.igem.org/mediawiki/2021/e/e1/T--SCU-China--rf5-1.jpg)

Figure 5.1. Behavior of TS over time in response to the following induction
scheme:aTc-Nothing-Ara-Nothing (NA mains inducer was removed)

5.2 Effect of different spacers on CRISPRi
efficiency

Since the TS
didn’t show the expected bistability phenomenon, which indicates that the efficiency of
CRISPRi which targeting the CDS region may be unsatisfactory. We designed three spacers
targeting the CDS regions for these two reporter genes at different locations,
respectively(Figure 5.2.1).

![](https://static.igem.org/mediawiki/2021/f/ff/T--SCU-China--results5.22.jpg
)

Figure 5.2.1 Binding sites of different spacers in the CDS region of reporter genes

A simple
NOR-gate was used to determine the change in expression intensity of different reporter
genes in the presence of only one inducer. After 10h of expression induction with 10ng/ml
aTc or 1% Ara at 37 degrees C, 120 μl of the bacterial solution was taken into a 96-well
plate and measured with an micro mplate reader to obtain its fluorescence intensity.

Interestingly, the spacer targeting the sfGFP CDS region had almost no effect on its
expression intensity, whereas the spacer targeting the mScarlet-I CDS region affected the
expression of sfGFP to some extent in addition to largely reducing the expression intensity
of mScarlet-I (Figure 5.2.2).

This
suggested that the orthogonality of our spacers targeting mScarlet-I is significantly low
and tends to trigger crosstalk between reporter genes of CRISPRi.

We considered
this and the extremely low efficiency of sfGFP CRISPRi as the main reasons why TS didn’t
work. The possible strategies are to target the promoter region to improve the efficiency of
CRISPRi or to use an artificially designed spacer to improve the orthogonality of these two
CRISPRi.

The former
approach proved to be feasible when we constructed iFFL.

![](https://static.igem.org/mediawiki/2021/d/da/T--SCU-China--rf5-3.jpg
)

Figure 5.2.2 CRISPRi efficiency of different spacers. The spacers we chose in construction of
the Toggle Switch were mScarlet-Spacer2 and sfGFP-spacer3.

..

6. Build iFFL by dCasΦ

Because iFFL can decouple the copy number and gene expression, and the gene expression will be in the same level. it is suitable for Vibrio natriegens who is sensitive to burden from gene circuit, because we can integrate circuit into genome and use iFFL to increase its performance

We chose three PAMs between -35box and -10box in p19 (Figure 6.1A), and perform CRISPRi, which showed a fantasy inhibition(Figure 6.1B).

![](https://static.igem.org/mediawiki/2021/8/8b/T--SCU-China--6.1.png
)

Figure 6.1

Then, we constructed plasmids which has dCasΦ-P19-mSarlet-P15AORI/PMB1ORI, and the colonies shows a successful iFFL (the same expression level of mSarlet).(Figure 6.2)

![](https://static.igem.org/mediawiki/2021/7/7a/T--SCU-China--6.2.png
)

Figure 6.2

## *Reference*

* 1. Takino, Junya et al. “Elucidation of biosynthetic pathway of a plant hormone abscisic acid in
  phytopathogenic fungi.” Bioscience, biotechnology, and biochemistry vol. 83,9 (2019): 1642-1649.
* 2. Beekwilder, Jules et al. “Polycistronic expression of a β-carotene biosynthetic pathway in
  Saccharomyces cerevisiae coupled to β-ionone production.” Journal of biotechnology vol. 192 Pt B
  (2014): 383-92.
* 3. Krebs, Jocelyn E., Elliott S. Goldstein, and Stephen T. Kilpatrick. Lewin's genes X. Jones
  &
  Bartlett Publishers, 2009.
* 4. Ro, Dae-Kyun, et al. "Production of the antimalarial drug precursor artemisinic acid in
  engineered yeast." Nature 440.7086 (2006): 940-943.
* 5. Wu, Tao, et al. "Engineering Saccharomyces cerevisiae for the production of the valuable
  monoterpene ester geranyl acetate." Microbial cell factories 17.1 (2018): 1-10.
* 6. Liu, Jianghua, et al. "Heterologous Biosynthesis of the fungal sesquiterpene trichodermol in
  Saccharomyces cerevisiae." Frontiers in microbiology 9 (2018): 1773.
* 7. Qi, Lei et al. “RNA processing enables predictable programming of gene expression.” Nature
  biotechnology vol. 30,10 (2012): 1002-6.
* 8. Souza-Moreira, Tatiana M et al. “Screening of 2A peptides for polycistronic gene expression
  in
  yeast.” FEMS yeast research vol. 18,5 (2018): 10.1093.
* 9. Ferreira, Raphael et al. “Multiplexed CRISPR/Cas9 Genome Editing and Gene Regulation Using
  Csy4
  in Saccharomyces cerevisiae.” ACS synthetic biology vol. 7,1 (2018): 10-15.
* 10. https://www.acs.org/content/acs/en/molecule-of-the-week/archive/p/phaseic-acid.html
* 11. Hou, Sheng Tao, et al. "Phaseic acid, an endogenous and reversible inhibitor of glutamate
  receptors in mouse brain." Journal of Biological Chemistry 291.53 (2016): 27007-27022.
* 12. Desjardins, Yves, et al. "Maple sap and syrup are a rich source of abscisic acid with
  potential
  benefits to health." XXVIII International Horticultural Congress on Science and Horticulture for
  People (IHC2010): International Symposium on 939. 2010.
* 13. Hou, Sheng Tao, et al. "Phaseic acid, an endogenous and reversible inhibitor of glutamate
  receptors in mouse brain." Journal of Biological Chemistry 291.53 (2016): 27007-27022.
* 14. Saito, Shigeki, et al. "Arabidopsis CYP707As encode (+)-abscisic acid 8′-hydroxylase, a key
  enzyme in the oxidative catabolism of abscisic acid." Plant physiology 134.4 (2004): 1439-1449.
* 15. Borchardt, Erin K et al. "Controlling mRNA stability and translation with the CRISPR
  endoribonuclease Csy4." RNA (New York, N.Y.) vol. 21,11 (2015): 1921-30.
* 16. Gu, Meigang et al. “Structure of the Saccharomyces cerevisiae Cet1-Ceg1 mRNA capping
  apparatus.”
  Structure (London, England : 1993) vol. 18,2 (2010): 216-27.
* 17. Takagi, T et al. “An RNA 5'-triphosphatase related to the protein tyrosine phosphatases.”
  Cell
  vol. 89,6 (1997): 867-73.
* 18. Mo, Jing et al. “TRADES: Targeted RNA Demethylation by SunTag System.” Advanced science
  (Weinheim, Baden-Wurttemberg, Germany) vol. 7,19 2001402. 16 Aug. 2020.
* 19. Fuchs, Anna-Lisa et al. “A general method for rapid and cost-efficient large-scale
  production of
  5' capped RNA.” RNA (New York, N.Y.) vol. 22,9 (2016): 1454-66.
* 20. Muttach, Fabian et al. “Synthetic mRNA capping.” Beilstein journal of organic chemistry vol.
  13
  2819-2832. 20 Dec. 2017.
* 21. Bouvet, Mickaël et al. “Stratégies de formation de la structure coiffe chez les virus à ARN”
  [Capping strategies in RNA viruses]. Medecine sciences : M/S vol. 28,4 (2012): 423-9.