iGEM 2019 — Team Aix-Marseille

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# Team:Aix-Marseille/Notebook

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[TEAM](https://2019.igem.org/Team%3AAix-Marseille/Team)

PROJECT

[Description](https://2019.igem.org/Team%3AAix-Marseille/Description)
[Design](https://2019.igem.org/Team%3AAix-Marseille/Design)
[Experiments](https://2019.igem.org/Team%3AAix-Marseille/Experiments)
[Notebook](https://2019.igem.org/Team%3AAix-Marseille/Notebook)
[Contribution](https://2019.igem.org/Team%3AAix-Marseille/Contribution)
[Results](https://2019.igem.org/Team%3AAix-Marseille/Results)
[Model](https://2019.igem.org/Team%3AAix-Marseille/Model)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AAix-Marseille/Parts)
[Basic Parts](https://2019.igem.org/Team%3AAix-Marseille/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AAix-Marseille/Composite_Part)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3AAix-Marseille/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AAix-Marseille/Public_Engagement)
[Collaborations](https://2019.igem.org/Team%3AAix-Marseille/Collaborations)
[News](https://2019.igem.org/Team%3AAix-Marseille/News)

[SAFETY](https://2019.igem.org/Team%3AAix-Marseille/Safety)

ACKNOWLEDGEMENTS

[Attributions](https://2019.igem.org/Team%3AAix-Marseille/Attributions)
[Sponsors](https://2019.igem.org/Team%3AAix-Marseille/Sponsors)

[MEDAL CRITERIA](https://2019.igem.org/Team%3AAix-Marseille/Medal_criteria)
[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Aix-Marseille)

Weekly meetings notebook

Week 1: 4th February - 8th February

It was the first meeting in the CNRS of Marseille for the presentation of the iGEM competition by James and 2018 team members.

Week 2: 11th February - 15th February

We attended a new presentation, more precise, of the competition and of its course. 2018 AMU team members developed their works by attribution, communication, funding, Human practices, modelisation and wetlab.

Week 3: 18th February - 22th February

We began to suggest some project ideas. Propositions were numerous and dealt with various field. Recurring themes revolved about Health and Environment issues. We decided to further reading about these by groups.

Week 4: 25th February - 1st March

15 subjects were pre-selected and we had to continue finding and working on bibliography. Besides, new members began to decide on what comities they wanted to take part (communication, finance, human practices, wetlab...).

Week 5: 4th March - 8th March

We continued to do more researches to find the perfect project to the AMU iGEM team. Besides, we began to fill in funding files and to find out about public events in Marseille.

Week 6: 11th March - 15th March

It was the D day! We had to choose our final subject of project. Among all of the ideas, two was selected to a final vote; the first one was developing a detection kit to diagnose Tuberculosis and the second one was finding a bioprocess to degrade lithium battery. With a margin of two votes, the final decision was the tuberculosis idea.

Week 7: 18th March - 22th March

First of all, Dr Stephane Canaan, a specialist of ***Mycobacterium tuberculosis***, made to us a presentation of the disease and gave to us some tracks to develop a detection kit. Then, we met "Semioz design graphique". He talked to us about some regulation for the logo in France, about design tools and presented to us the work ok Eiko Ojala, an Estonian illustrator and graphic designer. He thought that we should inspire us with this work to design our logo.

Week 8: 25th March - 29th March

There was a first global idea about the logo: one healthy lung and the other one sicked. We could represent that in different colors. The human practices community realized the first survey about typical knowledge of people about tuberculosis. The financial community finished and sent the first financial files. Finally, we created our logo (we finished the meeting very late).

Week 9: 1st April - 5th April

It was the day when we decided our name project. We were looking for a funny one. We voted for “I want to be free” in reference to the famous song “I want to be free” interpreted by the band Queen.

Week 10: 15th April - 20th April

In order to find the technic to develop our detection kit, we read lot of articles to discuss about which one we could use.

Week 11: 6th May - 11th May

In order to find the technic to develop our detection kit, we read lot of articles to discuss We received the famous iGEM kit. We did a skype with the [Thessaly Greek team](https://2019.igem.org/Team%3AThessaly). During this call, we talked about Tuberculosis, about our different project and why our two teams have chosen to work on this disease.
We sent to them our first survey to share around them.

Week 12: 27th May - 31th May

We discussed our first meeting with the public which takes part the weekend before: the “Souk des sciences”. It allowed us to meet an old members of the iGEM team of Lyon but also to meet people working with tuberculosis-infected patients. We talked about the things we can improve as an explanation of the tuberculosis test for diagnosis and the survey for the public.

Week 13: 11th June - 14th June

We announce on our social network the beginning of our crowdfunding campaign and we start the organization of our Meet-up.

Week 14: 17th June - 21th June

We start to discuss about the dry Lab.

Week 15: 27th June - 28th June

The collaboration with RAL Diagnostic is formalized and we are waiting for the equipment they sent to us. Moreover, we wrote the safety form and the project resum.

Week 28: 12th August - 16th August

We discuss about the advance of our experimentations. We received the Pins with our logo.

Week 29: 19th August - 23th August

The characterization of the chromoprotein is in progress, the construction of the TALE is done. We did a partnership with Wiloo.

Week 30: 26th August - 30th August

We had some problem in the wet Lab (the sonication breaks the TALE, the clonage of the polymerase seems to be impossible). We ordered the tee-shirts of the team.

Week 31: 9th September - 13th September

We made a return on the interview of Yannis and Jonnas for the Provence. We booked the flights to Boston and we worked on the design of the poster.

Week 32: 30th September - 4th October

We discussed about the “Forum des Biotech” which take place in Polytech and where we did a presentation of the iGEM contest and of our project.

Wetlab notebook

Week 16: 3rd June - 7th June

We set up the laboratory and installed the machines.

### Methylases

Bibliographic research to try to find a solution to capture DNA specifically without damaging it.

### Design of primers for PCR and PSR

Like many other isothermal amplification methods, the PSR method relies on the use of a DNA polymerase with strand displacement activity. Two primers with inverted sequences at their 5 'end are required to initiate the reaction. These inverted sequences were extracted from a botanical gene. The primers were developed with the aim of specifically targeting the Kat g and Rpob genes in Mycobacterium tuberculosis and Mycobacterium smegmatis.

The primers were designed based on the alignment of several published sequences of the Rpob and Katg gene (access numbers in GenBank are indicated in parentheses) in ***Mycobacterium smegmatis*** (CP001663.1) and ***Mycobacterium tuberculosis*** H37Rv (NC\_000962.3 ) as well as different outgroup species and then the identification of two regions with a high degree of homology surrounding a more variable region using the ClustaIW alignment tool of the Bioedit software (BioEdit v7.0.5, Tom Hall Ibis Therapeutics ). The physicochemical characteristics of the different primers were established using the Primer3plus software (Primer3PlusvVersion: 2.4.2) while the specificity of the different primer pairs was evaluated in silico using the Primer-Blast software against the base of data nr / nt.

### Test Strips

We performed all Biobricks we needed to carry out our experiment.
We build three antifreeze, polymerase, TolA3, PIII-N1-M13, and specific methylase proteins, which will be used to detect the specific gen of ***M.tuberculosis*** and the human amylase.

### TALs

We had a first meeting to establish the project's workflow.
Bibliography: On how to design the RNA guide of Crispr Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9).

Week 17: 10th June - 14th June

### Methylases

Bibliography on methylase at the end of the week thanks to MIT hacking paris

### PSR

The polymerases used for PSR must have a 5 '→ 3' DNA polymerase activity as well as a high strand displacement capacity, but must be devoid of 5 '→ 3' exonuclease function. For this we have firstly recover the sequences of ***Geobacillus stearothermophilus*** (CP008934.1) and ***Geobacillus thermodenitrificans*** (NC\_009328.1) which have a high activity around 60° C and a strong strand displacement capacity. However, in order to eliminate their 5 '→ 3' exonuclease function, we aligned these sequences with the large ***E. coli*** klenow fragment which is a polymerase devoid of exonuclease activity, and then recover only the part that aligns.

We then added a prefix and a suffix to the sequence in order to be able to insert it in the Rfc vector 25 as well as a 6-his tag and a TEV sequence to be able to purify it.

### Test Trips

We finished the bobriocks design and we moved in a new Lab and met all over research teams around us.

### TALs

Second meeting
Bibliography: On transcription activator-like (TAL) effectors

Week 18: 17 June - 21 June

Our internal wiki is working, we begin to fill the notebook and all pages!

### Lab

Cleaned-up the lab and unboxing all the material that Amidex financed.

### Methylases

Comparison of the different methylases according to the DNA used during the PSR. To select the methylases, we composed via SnapGene the enzymes of human amyloid (chosen for PSR) and the mycobacterium sequences (smegmatis and tuberculosis). Then as the choice of enzymes was limited, I used the NEB Cutter V2.0 software. After a while, my choice is turned on HhaI-M for mycobacterium and NlaIII-M for amylase. We then optimized the sequences using IDT.

##### PSR

**DNA extraction:**

Total DNA was extracted using the NucleoSpin Microbial DNA Kit (ref: 740235.50) from a 10 ml of ***Mycobacterium Smegmatis*** culture. The extracted DNA was eluted with 100 μl of elution buffer and the DNA was stored at -20° C.

**Polymerase Chain Reaction:**

In order to test the specificity of our primers on Mycobacterium smegmatis, we performed a PCR.

The polymerase chain reactions (PCR) were performed in a SENSOQUEST automated thermal cycler in 25 μL of PCR mix containing 12.5 μl of Econo taq PLUS GREEN 2X Master mix and 0.1 μl of each primer (10 μM). The 16S rRNA gene was amplified using the pair of primers described in the previous part (IDT) and 1 μL of DNA template (11 ng / μl). An initial denaturation step of 5 min at 95 ° C was followed by 39 denaturation cycles of 1 min each at 95 ° C, a hybridization step of 30s at 55 ° C and a treatment of 60 s of elongation at 72 ° C. Holding at 72 ° C for 5 minutes allowed complete extension of the PCR products. Negative controls consisting of PCR mixing without DNA template were included in each run of PCR..

For the Katg gene, we obtained a band around 300 bp which corresponded well to the expected result (theoretical size 304 bp).

For the Rpob gene, we obtained two bands, one at 600 bp and another 800 bp. The expected size of the amplicon was 600 bp. So we have a nonspecific band. In addition we observed strong bands of small sizes corresponding to the primers. This means that our primers are too concentrated.

**Polymerase Chain Reaction:**

In order to increase the specificity of our primer pair for the Rpob gene, we performed a new dilution from the stock solution of our primers, then we carried out a gradient PCR with the following temperatures: 48.8 ° C, 52.8, 57.2, 65 ° C. The protocols used are the same as those used previously.

**Results:**

We obtained a single band at 600 bp for the following temperatures: 48.8 ° C, 52.8, 57.2. Our primers have therefore amplified specifically the Rpob gene.

No bands were observed at 65° C.

##### Production of Dna Polymerases

We did a ligation in a pSB1K3 plasmid at room temperature for 3 hours, and transformed the product into DH5α competent cells.
We ran a colony-PCR.
Verification on an agarose gel of the colony-PCR (ligation with our insert and psB1K3 plasmid).
We launched starters on the confirmed positive colonies for the next day.
We purified the DNA (starter) using a DNA purification miniprep kit, measured the DNA
concentration on the NanoDrop and ran a digestion test and sent the product for sequencing.

Week 19: 24th June - 27th June

### Methylases

Command of the methylases.

### Polymerase spiral Reaction

The PSR reactions were carried out in a 25 μl reaction mixture containing the following components: 1.0 μl *Bst* DNA polymerase large fragment (New England Biolabs), 2.5 μl 10 × ThermolPol reaction buffer (New England Biolabs, including 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Tween 20), 1 ul Betain 0.8 M,1 ul MgSO4 6 mM, 3,5 10 mM each deoxynucleoside triphosphate. The amount of primers needed for one reaction was 1 ulfor both Forward and Reverse for Rpob and Kat g. Finally, 1 ul of DNA template was added to the reaction tube. The PSR reaction was carried out for 60 minutes at 57°C, 61°C, 64°C, 68°C.

The results were visualized by electrophoresis as explained previously.

**Results:**

No band could be obtained. This negative result is explained by the fact that amplicons obtained by the PSR are much too big and can not be visualized on gel.

#### Lab

Preparation of SDS 10% and APS 10%: that will be used to make Western-blot and SDS-PAGE gels.

**Polymerase spiral Reaction:**

In order to evaluate the PSR under different conditions, we added 1ul of SYBR GREEN in our mix. The results were visualized using the CFX 96 Real Time Cycler thermocycler by measuring the fluorescence every minute.

We tested different DNA concentrations (10 ^ 5 to 10 ^ 2 copies / ul) and different temperatures (66° C, 63.8° C, 59.6° C and 58° C)

**Results:**

The best results were obtained for concentrations of 10 ^ 4 and 10 ^ 3 of DNA at 63.1 ° C and 59.6 ° C. However, the amplification was not optimal, because the fluorescence did not even increase by a factor of 2.

**Polymerase spiral Reaction:**

The PSR was carried out under the same conditions as above with the target Katg gene because it contains a restriction site of the enzyme KatG. Thus, after amplifications a band should in theory be observable by gel electrophoresis. We therefore digested the amplification products with Eco RI, however no band was observed.

#### Test Trips

**CBD**

Extraction of te CBD DNA into the Kit Plate. DNA purification and enzymatic digestion was performed into the plasmid. The plasmidaws used to do a PCR and a clean up, and the optic density was measured thanks to the Nannodrop , either a DNA concentration 254 ng/µl.

#### TALs Biobrick

Digestion of pSB1K3 with EcoRI and PstI, and then a DNA clean up with Monarch kit after digestion.
We received the gene sequence from IDT containing the prefix and suffix.
PCR-amplification and agarose gel electrophoresis of the two linear TALEs received from IDT.

#### Antifreeze protein

**Polymerase chain reaction:**

The PCR was used to amplify antifreeze Gblocks, we're using three proteins in order to determine which one will work better in different conditions. After the amplification a band appears on every sample by gel electrophoresis.
A digestion of these inserts and T7\_RBS vector, was done using restriction enzymes.

**Ligation and transformation:**
anti-freeze inserts were added to T7-RBS vector, using a ligase. after the ligation, the vector was introduced into E.coli 5DH∂ using a transformation.
Cultures were incubated over night

#### TALs Biobrick

We ran a gradient-PCR to find the optimal primers hybridization temperature and verified it on an agarose gel.

[![T--Aix-Marseille--TALs PCR gradient picture.jpg](/wiki/images/d/de/T--Aix-Marseille--TALs_PCR_gradient_picture.jpg)](/File%3AT--Aix-Marseille--TALs_PCR_gradient_picture.jpg)

**Polymerase spiral Reaction:**

So far all our results have been negative, we think this is due to the concentration of betain. So we started again the mother solution of betain to 10M. Moreover we have inserted in our manipulations a positive control resulting from an article. We used a 0.8M betain concentration, a DNA concentration of 10 3 copies and the reaction was carried out at 65 ° C for 70 min. In order to visualize a possible amplification, 1 μl of SYBR GREEN (1/20) was added.

**Result**

For the negative control (mix without DNA) no color change was observed. For the positive control, we observe a high intensity green color which means that the PSR has functioned. For our samples (rpoB) a slight color change was observed which means that the amplification was not optimal.

### Test Strips

**Blue Green Chromoprotein**

A PCR on the Blue-Green Chromoprotein DNA fragment was realised to amplify the sample that we received by IDT. We verifyed the PCR product by Agarose gel elecrophoresis. The migration was performed during 25 minutes at 135 Volts, and was revelated with REDGel (BET), and the DNA concentration have been determined.

#### TALs Biobrick

Digested the gene with respective enzymes EcoRI and PstI, and then a DNA clean up with Monarch kit after digestion.

### Design of primers for PCR and PSR

The PCR was used to amplify Polymerase Gblocks, we are using two proteins Geobacillus stearothermophilus (CP008934.1) and ***Geobacillus thermodenitrificans***, we received the gene sequence from IDT.
We ran a gradient-PCR to find the optimal primers hybridization temperature and verified it on an agarose gel.
We have digested the gene with respective enzymes EcoRI and PstI, and then a DNA clean up with Monarch
kit after digestion.
We did a ligation in a pSB1K3 plasmid at room temperature for 3 hours, and transformed the product into DH5α competent cells. We ran a colony-PCR.
Then we have made a verification on an agarose gel of the colony-PCR (ligation with our insert and psB1K3 plasmid). We had no band at the right size.

Week 20: 1st July - 5th July

### Methylases

Recovery of genes blocks by IDT, the sequences sent on twist took too long to arrive.

### TALs Biobrick

We did a ligation in a pSB1K3 plasmid at room temperature for 3 hours, and transformed the product into DH5α competent cells.

### Test strips

Amplification of the p3-N1, TolA3-CBD and Blue green protein DNA fragment was performed. An agarose gel electrophoresis have been made to verifiyed the insert size.

[File:T--Aix-Marseille-- TolA3 and P3-N1 PCR amplification picture.jpg](/File%3AT--Aix-Marseille--_TolA3_and_P3-N1_PCR_amplification_picture.jpg "File:T--Aix-Marseille-- TolA3 and P3-N1 PCR amplification picture.jpg")

### TALs Biobrick

We ran a colony-PCR on the positive (white)/negative (pink) colonies.

### Test strips

An second PCR have been made in order to add a linker on each DNA fragment. That PCR was verified by agarose gel electrophoresis at 1%. We done a clean up on each DNA sample we obtained.

### TALs Biobrick

-Verification on an agarose gel of the colony-PCR (ligation with our insert and psB1K3 plasmid). We launched starters on the confirmed positive colonies for the next day.

-Digestion of the plasmid containing the promoter T7 and RBS (BBa\_K525998) with respective enzymes SpeI and PstI and both TALs but with XbaI and PstI enzymes, then we did a DNA clean up with Monarch kit after digestion. To finish we ran an overnight ligation in a psB1C3 plasmid at 16°C.

### Test Trips

**Fusion proteins constructions**

A plasmid digestion was performed in order to insert each DNA fragment into a vector. The PCR products containing each target DNA fragment have been cleaned by DNA clean up Monarch Kit.

A SLIC methode have been performed to insert two DNA fragment into a vector in order to create a fusion protein.

5µl of each sample was used to carry out a transformation with DH5α.

### **Anti-freeze result**

No bacteria survived. same digestion and ligation were preformed, but with a different mix

### Specificity of the PSR Assay

SYBR Greenı can bind to double-stranded DNA, emitting green fluorescence under UV light. Experiments were also performed to determine the detection of rpoB PSR assay when SYBR Green was used to enable observation of positive reactions using a visual color change

The PSR assay specificity for ***M.smegmatis*** detection was evaluated by SYBR GREEN indicator colorimetric assay. the strains tested are : ***Vibrio cholerae***, ***L..lactis***, ***Lysteria monocytogenes***, ***Burkholderia cenocepacia***, ***Acinetobacter baumannii***, ***E.coli 0157 H7***, ***Salmonella typhimurium***, ***Xenorhabdus nematophila***, ***Caulobacter crescentus***, ***Corynebacterium olae***, ***Photorhabdus luminescens***, ***Xanthomonas citri***, ***Streptococcus mutans***, ***M. smegmati***s (C+) and Water (C-).

A color change was visualized for ***M.smegmatis***, ***Lysteria monocytogenes*** and ***Burkholderia cenocepacia***.

### Sensitivity of PSR Assay

To compare the sensitivities of the PSR method and conventional PCR amplification, we conducted 10-fold serial dilutions of ***M.smegmatis*** genomic DNA (1.1ng/ul - 0,00011ng/ul) and subjected them to sensitivity testing. We tested primer for Rpob and Kat g.The detection limit for PCR was 0,11 ng/ul based on electrophoresis for rpoB and KatG gene.

### TALs Biobrick

* We purified the DNA from a confirmed positive colony-PCR using a DNA purification miniprep kit and measured the DNA concentration on the NanoDrop and ran a digestion test.
* The ligation in psB1C3 was then transformed the product into DH5α competent cells.

### Test Trips

**Fusion proteins constructions**

A screen PCR was realised to make sure that the TolA3-CBD and P3-N1-Green chromoprotein have inserted themselves into the plasmid. The results are shown in the following figure.(picture p24)

[File:T--Aix-Marseille-- TolA3 and P3-N1 PCR amplification 2 picture.jpg](/File%3AT--Aix-Marseille--_TolA3_and_P3-N1_PCR_amplification_2_picture.jpg "File:T--Aix-Marseille-- TolA3 and P3-N1 PCR amplification 2 picture.jpg")

Recovery of strain of ***mycobacteria*** previously denatured at 90° C for 30 minutes: ***M. tuberculosis*** ***MC26230***, ***M. avinum***, ***M. BCG*** and ***M. marinum***.

### **TALs Biobrick**

We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies, the amplification didn’t work.

### Test Trips

**Fusion proteins constructions**

A culture was performed on 3 colony of P3-N1-Green chromoprotein and 2 clones of TolA3-CBD.

The DNA sample from each selected clone were prepared by miniprep monarch kit and were sent to the sequencing.

Week 21: 8 July - 12 July

### Methylases

PCR to bind the methylase to the CBD.

### Anti-freeze result

Transformation still not working. After reviewing old results, the amount of plasmid is changed.

### TALs Biobrick

* We did an interne digestion on the purified colonies because the size of our insert is the same size of the mRFP of the plasmid psB1K3. Digestion on the purified sample with EcorI and NcoI from NEB, then was verified on an agarose gel and the results were negative.
* We recovered the transformation plates of the day 04.07 and ran another colony-PCR. We then launched starters from the confirmed positive colonies by an agarose gel.

### Test trips

**Fusion proteins constructions**

The sequnecing results shown that the inserts TolA3-CBD and P3-N1-Green chromoprotein were absents into the plasmids.

A plasmid digestion (pSB1K3) was made with EcoRI and PstI restriction enzyme.

DNA clean up by monarch kit.

### Anti-freeze result

After digestion and ligation, the culture is put into a transformation overnight

### TALs Biobrick

We purified the DNA (starter) using a DNA purification miniprep kit, measured the DNA concentration on the NanoDrop and ran a digestion test and sent the product for sequencing.

### Test trips

**Fusion proteins constructions**

A plasmid digestion (pSB1C3) was made with EcoRI and PstI restriction enzyme.

DNA clean up by monach kit.

Digestion of the PCR produts after the clean up with the T4 DNA polymerase: The T4 DNA polymerase was used to digerate the inserts (TolA3-CBD and P3-N1-Green chromoprotein) and the vector pSB1C3 during 2.30 minutes. The sample were introduced into ice for 10 minutes to stop the reaction. Transformation with DH5α strain cell.

### Anti-freeze result

A screenPCR was used to verify the size of the plasmid to make sure that the plasmid is well ligated to the insert. And a culture was incubated overnight

### TALs Biobrick

We recovered the transformation plates of the day 04.07 and ran another colony-PCR because it worked for only one TALE and verified it on an agarose gel.

### Test trips

**Fusion proteins constructions**

A scren PCR was realised in order to verified the incorporation of the inserts into the plasmid.

The DNA sample from each selected clone were prepared by miniprep monarch kit and were sent to the sequencing.

### Anti-freeze result

The culture that shown the must adequate size was chosen to do a sequencing, so a starter that contains antibiotics (chloramphenicol) was used to incubate overnight.

### TALs Biobrick

We launched starters on the confirmed positive colonies for the next day.

### TALs Biobrick

* We purified the DNA from a confirmed positive colony using a DNA purification miniprep kit and sent the product for sequencing.
* Digestion of the plasmid (psB1C3) containing the promoter T7 and RBS ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) and our TALE gene with respective enzymes SpeI and PstI and both chromoprotein chosen but with XbaI and PstI enzymes, then we did a DNA clean up with Monarch kit after digestion, measured the DNA concentration on the NanoDrop and ran a digestion test.

Week 22: 15th July - 20th July

### Methylases

Slic to link CBD to the different methylases.

### Anti-freeze results

Mini-preps were performed to purify the plasmid. and sent to sequencing.

### TALs Biobrick

We ran a ligation in pSB1C3 at room temperature, was then transformed the product into DH5α competent cells (didn't worked).

### Test strips

Digestion of the plasmid (psB1C3) containing the promoter T7 and RBS ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) with PstI and PstI-HF a, then we did a DNA clean up with Monarch kit after digestion at 37°C. An hybridation have been made with the T4 DNA polymerase and a transformation with ***E.coli*** DH5α competent cells have been performed.

### TALs Biobrick

Digestion of the plasmid (psB1C3) containing the promoter T7 and RBS ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) with SpeI and PstI-HF and our TALE gene with XbaI and PstI, then we did a DNA clean up with Monarch kit after digestion, measured the DNA concentration on the NanoDrop and did a ligation overnight at 16°C.

### Test strips

We use the transformants DH5α to carry out a miniprep. First cwe inoculate these transformants into LB medium containing ampicilline at 37°C during 16 hours.

### TALs Biobrick

We cloned the gene in pSB1C3 overnight and trasnformed it into DH5α competent cells.

### Test strips

Miniprep

### TALs Biobrick

* We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies. We had non-conclusive results.
* We did digestion and ligation with a linear plasmid named psB1K3 with our gene overnight at 16°C.
* We did another PCR-amplification and agarose gel electrophoresis of the two linear gene received from IDT, to renew our DNA stocks.

### TALs Biobrick

We cloned the gene in pSB1K3 overnight and trasnformed it into DH5α competent cells.

### Test strips

We use the plasmids that are purified during the miniprep step to do PCR in order to add a linker on each gene fragments.

Clean up PCR.

SLIC : We used Cut plasmid (pSB1K and PSB1A3) to carry out a SLIC. A ration of 1 vector for two inserts have been calculated for each differents plasmids we used. An Hybridation have been made with the T4 DNA polymerase. The mix have been incubated at room temperature during 2.30 minutes and have been placed into ice for 10 minutes to stop the reaction. The transformation of 50µl of DH5α competents cells with 5µl of hybridation mix (insert/vector).

The culture have been incubate in a petri dish containing LB medium and Kanamycine or ampicilline antibiotics during a week-end on the a bench.

We ran a ligation in pSB1C3 at room temperature, was then transformed the product into DH5α competent cells.

### Polymerase spiral reaction

Receipt of strains of denatured **mycobacteria** that have lost all pathogenicity.
DNA extraction was performed from a 1ml pellet using the Nucleo Spin Microbial DNA kit. The DNA concentration was measured by Nano Drop .
We performed a PCR to test our ***M. tuberculosis*** specific primers. and then we performed a PSR to test our ***M. tuberculosis*** specific primers.
To analyze the PSR reaction product, the product was purified with a DNA purification kit, anddigested with PvuI restriction enzyme cutting sites found in the original sequence
the digestion product has been visualized on agarose gel.

Week 23: 22th July - 27th July

### Methylases

Other week of SLICs to bind the methylase to the CBD.

### Anti-freeze result

The result of sequencing was incorrect (empty vector).
An other protocol was conducted (linear plasmid)

### TALs Biobrick

We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies. We had non-conclusive results.

### Test strips

A lot of clones was observed on the petri dish.

A screen PCR with one Taq mix was performed to verified the presence of the inserts into the plasmids.

Storage at 4°C.

The results of the screen PCR are shown below.

[![T--Aix-Marseille--Jade agarose gel electrophoresis p38 picture.png](/wiki/images/2/2a/T--Aix-Marseille--Jade_agarose_gel_electrophoresis_p38_picture.png)](/File%3AT--Aix-Marseille--Jade_agarose_gel_electrophoresis_p38_picture.png)

Culture over night of the clones 1,4, 12 and 18 of the transdormants containing the pSB1A3 plasmid with the CBD-TolA3 insert.

### TALs Biobrick

We digested the gene sequence with EcoRI and PstI for cloning in pSB1C3. We had the right band at the right size when migrated on an agarose gel then we cloned the digested gene sequence in pSB1C3 and transformed into DH5α competent cells.

### Test strips

A SLIC have been made with the ratio 1 :2 :2 (vector/insert 1/insert 2), then we incubate the hybridation mix at room temperature during 2.30 minutes with the T4 DNA polymerase. The reaction have been stoped when we put the sample into ice for 10 minutes.

Transformation of 50µL of competents cells by heat choc with 3 to 5 µL of the inserts/vector mix.

### TALs Biobrick

We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies. Afterward, we prepared 3 starters on the confirmed positive colonies for plasmid purification.

### Test strips

* Miniprep
* Verification of the presence of the insert into the plasmid by digestion with restriction enzymes .
* We digested the 3 clones containing TolA3-CBD ( clones 1,4 and 12) with two differents restriction enzymes such as EcoRI and PstI-HF. We carry out three differentes digestion for each sample. These digestions have been made during 1.30 hour at 37°C.
* The results are shown below

[![T--Aix-Marseille--Jade agarose gel electrophoresis p41 picture.png](/wiki/images/e/ee/T--Aix-Marseille--Jade_agarose_gel_electrophoresis_p41_picture.png)](/File%3AT--Aix-Marseille--Jade_agarose_gel_electrophoresis_p41_picture.png)

### TALs Biobrick

* We did a digestion of verification and we didn't had the right band at the right size it had a size of a mRFP
* A gel extraction was done using a Macherey-Nagel kit, which allowed us to extract only the plasmid without the mRFP.

### Test strips

* Digestion of pSB1A3-CBD-TolA3 with XbaI and pSB1-HF restriction enzyme. We digested CBD DNA fragment with XbaI and the TolA3 DNA fragment with PstI.
* Digestion of the plasmid pSB1C3 containing the T7RBS domain with SpeI and PstI-Hf restriction enzyme.
* DNA clean up by monarch kit
* Ligation : The T4 DNA ligase was used to ligate the insert TolA3-CBD and the T7RBS plasmid together. We put the T4 DNA ligase in contact with the insert/vector mix for 2 hours at room temperature.
* DNA clean up by monarch Kit
* Transformation of 50µL of competents cells by heat choc with 3 to 5 µL of the inserts/vector mix. Incubation at 37°C for 16 hours.

### Test strips

* Miniprep
* Sequencing
* PCR of the blue-green chromoprotein and the blue chromoprotein have been made in order to add a linker on each DNA fragment. These linker have complementary base.
* Clean up PCR .
* Storage at -20°C

##### Polymerase Spiral reaction

To test the detection limit of the test for ***M. tuberculosis***, we used genomic DNA from ***M. tuberculosis*** as a test object, and performed a series of 10-fold dilutions of the total DNA solution, generating in nucleic acids from 10^5 to 10^1 copies.

Week 24: 29th July - 3rd August

### Methylases

An other week of SLICs.

### TALs Biobrick

With the plasmid extracted we did a ligation for 2 hours at 25°C with our gene, that was then transformed into DH5α competent cells.

### Test strips

Two differents SLIC have been made with the ratio 1 :2 :2 (vector pSB1A3 linear plasmid) ( /insert 1 (Blue chromoprotein )/insert 2 ( P3-N1)) and (vector psB1K3 linear plasmid)/ insert 1 ( Blue-green chromoproetin/ insert 2 ( P3-N1)), then we incubate these hybridation mix at room temperature during 2.30 minutes with the T4 DNA polymerase. These reaction have been stoped when we put the samples into ice for 10 minutes.

Transformation of 50µL of competents cells by heat choc with 3 to 5 µL of both inserts/vector mix.

Incubate 16 hours at 37°C.

### TALs Biobrick

* We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies. We had no band at the right size.
* We used the SLIC/digestion method to clone our gene into a linear plasmid psB1K3, follow up with a transformation in DH5α competent cells.

### Test strips

* Miniprep
* A PCR colony have been made on each Blue Green –P3-N1 and Blue-P3-N1 clones.The results are shown below.
* Incubation af each clones at 37°C for 16 hours.

[![T--Aix-Marseille--agarose gel electrophoresis p45 picture.png](/wiki/images/8/85/T--Aix-Marseille--agarose_gel_electrophoresis_p45_picture.png)](/File%3AT--Aix-Marseille--agarose_gel_electrophoresis_p45_picture.png)

[![T--Aix-Marseille--agarose gel electrophoresis p46 picture.png](/wiki/images/7/76/T--Aix-Marseille--agarose_gel_electrophoresis_p46_picture.png)](/File%3AT--Aix-Marseille--agarose_gel_electrophoresis_p46_picture.png)

### TALs Biobrick

* We did an other PCR-amplification and agarose gel electrophoresis of the two linear gene received from IDT, to renew our DNA stocks and we did a digestion with XbaI and PstI to clone into a plasmid containing T7 RBS that was cut with SpeI and PstI enzymes.
* We recovered the transformation plates of the SLIC and ran a colony-PCR on the supposedly positive colonies, 1 of 2 genes had the correct band size so we launched a starter.

### Test strips

* Clean up PCR of the Blue-Green chromoprotein-P3-N1 and the Blue chromoprotein-P3-N1 have been made.
* Sequencing

### TALs Biobrick

* We sent the miniprep product for sequencing.
* We did a ligation of the digested gene sequence in pSB1C3 with T7 RBS and transformed into DH5α competent cells. (Julie)

### Test strips

Sequencing results : We have no insert between the prefix

### TALs Biobrick

We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies.

[![T--Aix-Marseille--TALs PCR COLO 2 picture.jpg](/wiki/images/2/26/T--Aix-Marseille--TALs_PCR_COLO_2_picture.jpg)](/File%3AT--Aix-Marseille--TALs_PCR_COLO_2_picture.jpg)

Week 25: 5th August - 9th August

### Methylases

Transformation, ligation and digestion of the methylases.

### TALs Biobrick

* We recovered the sequencing results that were positive (SLIC).
* We launched 6 starters of colonies that had the correct band size after PCR-Screen.

### TALs Biobrick

* We did a digestion of verification on the minipreps sample, then sent the minipreps product for sequencing.
* We did multiple transformation to renew our stock with the RED, Bleu-Green, Bleu chromoprotein and the the plasmid (psB1C3 T7 RBS) containing both of our genes of interest.

### TALs Biobrick

* We recovered the sequencing results that were positive (our gene + psB1C3 with T7 RBS)
* Digestion of the plasmid containing the promoter T7 and RBS ([BBa\_K525998](http://parts.igem.org/Part%3ABBa_K525998)) and our gene (TAL) with respective enzymes SpeI and PstI and both chromoproteins (Red ([BBa\_K592012](http://parts.igem.org/Part%3ABBa_K592012)) and Bleu ([BBa\_K1033928](http://parts.igem.org/Part%3ABBa_K1033928))) but with XbaI and PstI enzymes, then we did a DNA clean up with Monarch kit after digestion. To finish we did a ligation for 2 hours at 25°C and to finish they were then transformed into DH5α competent cells.

### TALs Biobrick

* We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies.
* We launched 5 starters of colonies that had the correct band size after PCR-Screen.

### Specificity PSR test

Realization of PSR with different DNA concentrations of ***M. tuberculosis*** for a test of sensitivity
The specificity of the PSR Assay for M.tuberculosis Primers was tested.

Week 26: 12th August - 17th August

### Methylases

Slics finally worked, but we lack of time.

### TALs Biobrick

* We sent the minipreps product for sequencing and launched a transformation in BL21 for the production.
* Digestion of the plasmid containing the promoter T7 and RBS ([BBa\_K1033928](http://parts.igem.org/Part%3ABBa_K1033928)) and our gene (TAL) with respective enzymes SpeI and PstI and cjBlue green chromoprotein ([BBa\_K592011](http://parts.igem.org/Part%3ABBa_K592011)) but with XbaI and PstI enzymes, then we did a DNA clean up with Monarch kit after digestion. To finish we did a ligation for 2 hours at 25°C and to finish they were then transformed into BL21 competent cells.
* AMY1 DNA was obtained from sputum. DNA extraction was performed from a pellet of 1ml of saliva using the Nucleo Spin Microbial DNA kit. We performed a PCR to test our AMY1 specific primers and then a PSR to test our AMY1 specific primers.

### TALs Biobrick

* We launched 3 starters of 10mL for the productions test.
* We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies and launched 1 starter of a colony that had the correct band size.

### TALs Biobrick

* We recovered the sequencing results that were positive, psB1C3 containing T7 RBS+ TAL (our gene)+ Chromoprotein
* We sent the minipreps product for sequencing
* We induced till an OD of 0,5/0,6 at 37°C with a rotation of 850rpm then we added IPTG and incubateur during 3h at 30°C. 1mL of the cell culture was used to check the production on SDS page, by incubating the cell culture for 5 min at 95°C.

Week 27: 19th August - 24th August

### TALs Biobrick

We launched 3 starters of 10mL for the productions.


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# Team:Aix-Marseille/Experiments

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Experiments

Polymerase spiral reaction (PSR)

## Materials and methods

#### Why *M. smegmatis* ?

***M. smegmatis*** is commonly used in Mycobacteria research because of its important growth rate. The time and heavy infrastructure needed to work with pathogenic species have prompted researchers to use ***M. smegmatis*** as a model for mycobacterial species. They share more than 2 000 homologous genes with ***M. tuberculosis*** and share the same wall structure cell. These many properties in common have pushed us to carry out our work on ***M. smegmatis***. Once we master the PSR, we'll run the tests on ***M. tuberculosis*** cultures provided by [Dr. CANAAN](https://2019.igem.org/Team%3AAix-Marseille/Interviews) for [safety](https://2019.igem.org/Team%3AAix-Marseille/Safety).

## Primers design

The PSR primers were designed based on the alignment of four published sequences of the rpoB gene of ***M. tuberculosis*** ([Genbank](https://www.ncbi.nlm.nih.gov/genbank/) access numbers: MG995113.1, MG995115.1, MG99511.1 and MG99511.1) and the identification of two regions with high degree homology framing the region where the mutation responsible for acquiring resistance to Rifampicin is. It's done using [Clustalo](https://www.ebi.ac.uk/Tools/msa/clustalo/) mutiple alignment tool. The alignment file included various rpoB DNA sequences of various Mycobacteria species to select the most specific region for ***M. tuberculosis***.

Afterwards, an oligonucleotide sequence of exogenous origin, described by *Liu and al*, has been incorporated within the 5’ of each primer. We extracted the N and Nr sequences of a botanical gene to avoid a non-specific reaction with the target gene. The physicochemical characteristics of the different primers were established using the [Primer3Plus software](http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). While the specificity of the different primer sets has been evaluated *in silico* using the [Primer3Plus software](http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) against the nr/nt database. The primers designed for ***M. smegmatis*** were developed using the same procedure.

Alternatively, we used a primer set specific to ***M. tuberculosis*** suitable for ***M. smegmatis***: **Ft (5'-GTGCCCGCAAAGTGTGGCTAACTTGCGCGATGGCGAACTCA-3')** and **Bt (5' GTGCCCGCAAAGTGTGGCTAACTTAGTTTGGTCATCAGCCGTTC-3')** described by *Liuyu Huang et al*. These primers served as positive control. The primers designed for amy1 were designed with the exception of their specificity and their sensitivity because firstly they serve as the amplification positive control. The gene codes for the amylase protein. It's abundantly present in the sputum sample.

## DNA extraction

DNA was extracted from a 1 mL ***M. smegmatis*** culture. The measured DNA concentration by NanoDrop was 104 ng/µL. The ***M. tuberculosis*** DNA was provided by [Dr. CANAAN](https://2019.igem.org/Team%3AAix-Marseille/Interviews). The DNA extraction was carried out using a 1 mL pellet using the kit. The measured DNA concentration by NanoDrop was 81 ng/µL. amy1 DNA was extracted from a 1 mL sputum sample. All DNA extractions were done using the [NucleoSpin® Microbial DNA](https://www.mn-net.com/tabid/12620/default.aspx) kit.

## Amplification

PSR assay was carried out at a temperature of 62°C and a time span of 60 minutes. The reaction mix consisted of 1 uL forward and reverse PSR primers, 1.4 mM dNTPS (from Sigma-Aldrich), 0.6 M of Betaine, 4 mM MgSO4, 12 U of DNA polymerase large fragment (from New England Biolabs), 2.5 uL of 10X Thermopol reaction buffer (consisting of 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, and 0.1% Tween 20), 1ul of DNA template, and the volume was adjusted to 25 uL with nuclease-free water.

A typical positive PSR assay produced a multiple band scale on a colored agarose gel, resulting from a simultaneous extension of the DNA polymerase. To analyze the PSR reaction product, the product was purified with a DNA purification kit, and digested with the PvuI restriction enzyme cutting sites found in the original sequence. The result showed that the product was digested and formed a single fragment. At the same time, SYBR green has been added into the reaction mix. SYBR Green can bind to double-stranded DNA by emitting green fluorescence. After the end of the reaction, 1 uL of SYBR Green diluted 1:1000 was added to PSR products. A bright green color indicated positive amplification.

![T--Aix-Marseille--Amplification fig1 picture.png](/wiki/images/5/5e/T--Aix-Marseille--Amplification_fig1_picture.png)

Figure A: Agarose gel electrophoresis of PSR products (From left to right)
Well 1 : Molecular size marker.
Well 2 : PSR amplification products.
Well 3 : PSR amplification products digested by PvuI.

## Results

### Specificity test for *M. smegmatis*

To test the specificity of our test for *M. smegmatis* we used 18 bacteria: mycobacterium: vitro cholera, *Lactobacillus lactose*, *Listeria monocytogenes*, *Burkholderia cenocepacia*, *Acinetobacter baumannii*, *E.coli 0157 H7*, *Salmonella typhimurium*, *Xenorhabdus nematophila*, *Caulobacter crescentus*, *Corynebacterium olae*, *Photorhabdus luminescens*, *Xanthomanos citri*, *Streptococcus mutans*. Results are shown in the figure B. Positive results were obtained for *M. smegmatis*, *Listeria monocytogenes* and *Burkholderia cenocepacia*, demonstrating that our test had an average specificity for *M. smegmatis*.

![T--Aix-Marseille--Test de spécificité 1 picture.png](/wiki/images/e/e0/T--Aix-Marseille--Test_de_sp%C3%A9cificit%C3%A9_1_picture.png)

Figure B: Visual detection of negative and positive results of PSR amplification products
(1) ***Vibrio cholerae***,(2) ***Lactobacilus lactis***, (3) ***Lysteria monocytogenes***, (4) ***Burkholderia cenocepacia***, (5) ***Acinetobacter baumannii***,(6) ***E. coli 0157 H7***, (7) ***Salmonella typhimurium***,(8) ***Xenorhabdus nematophila***, (9) ***Caulobacter crescentus***, (10) ***Corynebacterium olae***, (11) ***Photorhabdus luminescens***, (12) ***Xanthomonas citri***, (13) ***Streptococcus mutans*** (14) Water (negative control), (15-16) ***M. smegmatis*** (Positive control).

### Detection limit for *M. smegmatis*

To test the detection of the test for the test for ***M. smegmatis***, we used genomic DNA of ***M. smegmatis*** as a test object, and performed a series of 10-fold dilutions of the solution of total DNA, generating nucleic acid concentrations of 10^5 to 10 copies. The test was made with distilled water as a negative control. The results, as shown in FIG, indicate that the detection limit (LD) was 10^2 copies/µl. The limit of detection was also measured by PCR for the same DNA concentrations. The PCR products were separated by agarose gel electrophoresis and stained with red gel. The gels were viewed using the Gel Doc system. The results, as shown in FIG. indicate that the detection limit was 10^2 copies/µl. The sensitivity of the PSR test for the detection of M.smegmatis was substantially equal to that
of the test of polymerase chain reaction, based on gel electrophoresis results, agarose and the color change detected with the naked eye.

![T--Aix-Marseille--Limite test smegmatis picture fig 3.png](/wiki/images/3/38/T--Aix-Marseille--Limite_test_smegmatis_picture_fig_3.png)

Figure C: Results of the limite of detection for Mycobacterium smegmatis
The nucleic acids concentrations into those tubes 1-5 was respectivly de 10^6< copy / µL, 10^5 copy / µL, 10^4 copy / µL, 10^3< copy/ µL, 10^2< copies / µL, 100 copies / µL. The tube 6 have been maintained as a negative control.

Figure D: Agarose gel elctrophoresis of PCR product.
Well 1 : Size Marker Smart ladder. Well 2 : 10^5 copies / µL. Well 3 : 10^4 copies / µL. Well 4 : 10^3 copies / µL Well 5 : 100 copies / µL. Well 6 :10 copies / µL. The Well 7 have been maintained as negative control.
Theoretical size : 598 bp.

![T--Aix-Marseille--electrophorèse fig 4 picture.png](/wiki/images/4/46/T--Aix-Marseille--electrophor%C3%A8se_fig_4_picture.png)

### Specificity test for *M. tuberculosis*

To test the specificity of our test for ***M. tuberculosis***, we used 19 bacteria *Vibrio cholerae* , *Lactobacilus lactis*, *Listeria monocytogenes*, *Burkholderia cenocepacia*, *Acinetobacter baumannii*, *E.coli 0157 H7*, *Salmonella typhimurium*, *Xenorhabdus nematophila*, *Caulobacter crescentus*, *Corynebacterium olae*, *Photorhabdus luminescens*, *Xanthomonas citri*, *Streptococcus mutans*, *Bacteroides fragiles*, *Mycobacterium avium*, *Mycobacterium marinum*, *Mycobacterium smegmatis*, a BCG strain, *Mycobacterium abscessus* and *M. tuberculosis* (positive control). We used water as a negative control.

Results are shown in the figures E and F. We have positive (green tubes) results with ***M. tuberculosis*** and the BCG strain.

![T--Aix-Marseille--Test de spécificité TB picture.png](/wiki/images/0/0b/T--Aix-Marseille--Test_de_sp%C3%A9cificit%C3%A9_TB_picture.png)

Figure E: Results of the specificity test of ***M. tuberculosis*** (1) ***Vibrio cholerae***,(2) ***Lactobacilus lactis***, (3) ***Lysteria monocytogenes***, (4) ***Burkholderia cenocepacia***, (5) ***Acinetobacter baumannii***,(6) ***E. coli 0157 H7***, (7) ***Salmonella typhimurium'***,(8) ***Xenorhabdus nematophila***, (9) ***Caulobacter crescentus***, (10) ***Corynebacterium olae***, (11) ***Photorhabdus luminescens***, (12) ***Xanthomonas citri***, (13) ***Streptococcus mutans***, (14) ***Bacteroides fragiles***, (15) Water (C-), (16) ***M. tuberculosis*** (C+).

Figure F: Specificity tests of ***M. tuberculosis*** (1) ***M. tuberculosis***, (2) BCG strain, (3) ***M. avium***, (4) ***M. marinum***, (5) ***M. smegmatis***,(6) ***M. abscessus***.

![T--Aix-Marseille--Test de spécificité TB 2 picture.png](/wiki/images/1/1c/T--Aix-Marseille--Test_de_sp%C3%A9cificit%C3%A9_TB_2_picture.png)

### Detection limit for *M. tuberculosis*

To test the detection limit of the procedure, we used the genomic DNA of *M. tuberculosis* as a test object, and performed a series of 10-fold dilutions the total DNA solutions. We generated nucleic acid concentrations of 10^5 to 10 copies. The test was made with distilled water as a negative control. The results as shown in Figure G. It indicates that the detection limit (LD) was 10^2 copies/µL. The limit of detection was also measured by PCR for the same DNA concentrations. The PCR products were separated by agarose gel electrophoresis and gel-stained red. The gels were visualized. The results, as shown in figure G indicate that the detection limit was 10^2 copies/µL.

![T--Aix-Marseille--Limite detection TB picture.png](/wiki/images/3/32/T--Aix-Marseille--Limite_detection_TB_picture.png)

Figure G: Detection limit test for *M. tuberculosis*.
The nucleic acids concentrations into the tube 1-5 were respectivly at 10^5 copies / µL, 10^4 copies / µL, 10^3 copies / µL, 10^2 copies / µL, 10^1 copies / µL. Tube 6 serve as a negative control.

Figure H: Agarose gel electrophoresis for PCR products.
(From left to right). Well n°1 : molecular marker. Well n°2 : 10^6 copies / µL. Well n°3 : 10^5 copies / µL. Well n°4 : 10^4 copies / µL Well n°5 :10^3 copies / µL. Well n°6 :10^2 copies / µL. Well n°7 : 10 copies / µL. The well n°8 serves as a negative control.
Theoretical size: 432 bp

![T--Aix-Marseille--gel agarose dectection TB picture.png](/wiki/images/a/ac/T--Aix-Marseille--gel_agarose_dectection_TB_picture.png)

## Amylase

Sputum amylase is a polypeptide chain of 511 amino acids and encoded by an the amy1 gene located on Human chromosome 1.

![T--Aix-Marseille--amylase test picture.png](/wiki/images/a/af/T--Aix-Marseille--amylase_test_picture.png)

Figure I: PSR test of the amy1 gene (1) Saliva DNA extract, (2) Water (Negative control)

#### References

* Development and application of a rapid Mycobacterium tuberculosis detection technique using polymerase spiral reaction. Wei Liu, Dayang Zou, Xiaoming He, Da Ao, Yuxin Su, Zhan Yang, Simo Huang, Qinghe Zhao, Yue Tang, Wen Ma, Yongfeng Lu, Jing Wang, Xinjing Wang & Liuyu Huang. Scientific Report.
* Polymerase Spiral Reaction (PSR): A novel isothermal nucleic acid amplification method. Wei Liu, Derong Dong, Zhan Yang, Dayang Zou, Zeliang Chen, Jing Yuan & Liuyu Huang. Scientific Report
* Polymerase spiral reaction (PSR): a novel, visual isothermal amplification method for detection of canine parvovirus 2 genomic DNA. Vikas Gupta,Soumendu Chakravarti, Vishal Chander, Saurabh Majumder,Shabir Ahmad Bhat, Vivek Kumar Gupta,Sukdeb Nandi
* Rapid detection of Pseudomonas aeruginosa targeting the toxA gene in intensive care unit patients from Beijing, China. Derong Dong, Dayang Zou, Hui Liu, Zhan Yang, Simo Huang, Ningwei Liu, Xiaoming He, Wei Liu and Liuyu Huang1
* Helix loop-mediated isothermal amplification of nucleic acids. Rui Mao, ab Lifei Qi,ab Zhuo Wang, Hongtao Liu ac and Yuguang Du.
* Rapid visual isothermal nucleic acid-based detection assay of Brucella species by polymerase spiral reaction. A. Das , B. Kumar, S. Chakravarti , C. Prakash , R.P. Singh, V. Gupta , K.P. Singh , R.K. Agrawal , V.K. Chaturvedi , Abhishek and G. Shrinet
* Novel polymerase spiral reaction assay for the visible molecular detection of porcine circovirus type 3. Jun Ji , Xin Xu , Xueyu Wang, Kejing Zuo , Zhili Li , Chaoliang Leng , Yunchao Kan , Lunguang Yao and Yingzuo Bi
* Takahashi, T. et al. Nested polymerase chain reaction for assessing the clinical course of tuberculous meningitis. Neurology. 64, 1789–1793 (2005).
* Rapid molecular detection of tuberculosis and rifampin resistance. Boehme CC1, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, Allen J, Tahirli R, Blakemore R, Rustomjee R, Milovic A, Jones M, O'Brien SM, Persing DH, Ruesch-Gerdes S, Gotuzzo E, Rodrigues C, Alland D, Perkins MD.
* Evaluation of the analytical performance of the Xpert MTB/RIF assay. Blakemore R1, Story E, Helb D, Kop J, Banada P, Owens MR, Chakravorty S, Jones M, Allan

TALs

### Transcription Activator-Like (TALs)

We used the TALs proteins and linked them to a chromoprotein. The complex recognizes the PSR product. The complex is the main detection component of our procedure.

### TAL2 production

We were able to insert the TAL2 gene into a plasmid containing the T7 promoter and RBS which is a psB1C3 vector. SDS-PAGE was performed and stained with Coomassie blue using cells containing this Biobrick, to verify the production of our protein we did different growing conditions to determine the best growing conditions by using two types *E. coli* bacteria :

* *E. coli K-12 DH5-alpha*
* BL21 DE3

Figure 1. SDS-PAGE of the production in *E. coli K-12 DH5-alpha* of TAL, well 1, 2 and 3 are not induced of TAL1, TAL2 clone1 and TAL2 clone2, respectively. Well 4, 5 and 6 are induced at 30°C with 0.1M IPTG of TAL1, TAL2 clone1 and TAL2 clone2, respectively.

![T--Aix-Marseille--TAL2 production picture.png](/wiki/images/a/a2/T--Aix-Marseille--TAL2_production_picture.png)

![T--Aix-Marseille--TALs production 1 picture.png](/wiki/images/5/5d/T--Aix-Marseille--TALs_production_1_picture.png)

Fig 2. SDS-PAGE of the production in BL21 DE3 of TAL. Well 1, 3 and 5 are induced at 30°C with 0.1M IPTG for 3 hours of TAL1, TAL2 clone1 and TAL2 clone2, respectively. Well 2,4 and 6 are induced at 16°C with 0.1M IPTG overnight.

We used 2 different strains of E. coli Dh5 alpha, and a BL21 strain. There are bands at approximatively 42kDa, that correspond to TAL2 molecular weight that was engineered.
After analyzing these results, the induction condition at 0.1M IPTG at 30 ° C is chosen for the rest of the experiments. As we can see wither our protein are amplify overnight or for 3hours theirs the same quantity of protein that are produced, and wither our protein were produced in BL21 DE3 or DH5a theirs also the same amount of protein that are produced.
So, we choose to use the proteins that was produced overnight in BL21 DE3.

### TAL2 purification

After having realized the TAL2 production tests, the purification was realized in order to obtain pure protein fractions. For this, the E. coli BL21 cells were lysates and then are purified. Purification of the protein was realized with a HisTag column which is a manual chromatography system for quick, easy ad small quantity purification. The fusion of the histine tag with TAL2 allows to separate our protein from the other proteins.
To confirm that the elution corresponds to the TAL2, a Western Blot of the fractions corresponding to the elution as well as the non-retention is realized (Fig.3). The deposit on the SDS-PAGE of the no-retained fraction allows to check if our has been retained on the column.

![T--Aix-Marseille--TAL2 WB purif picture.png](/wiki/images/8/82/T--Aix-Marseille--TAL2_WB_purif_picture.png)

Fig 3. Western Blot analysis of TAL2 after purification with HisTag column. LD= loading sample, NR= non-restraint, E1 to E5 correspond to the eluted fractions.

After Western Blot visualization (fig. 3), the histidine tag allows to purify our protein from the other proteins. Red Ponceau staining was use on this Western Blot, which conclude why the bands at E3, E4 and E5 are not intense, because Ponceau staining are specific to our protein. An intense band is observable approximately 42 kDa corresponding to TAL2. However, the 42kDa band is observable in the no-restrained, it means that there was too much protein and the used column saturated with our sample. After purification, a pool of fractions was done. Now that the protein is purified, an activity test is done to check if the TAL2 has a high affinity for the DNA amplify.

### TAL2 activity test

To verify the functionality of this part we did an electrophoretic mobility shift assay (EMSA) also none as a gel shift assay, which is a method that’s able to analyze the potential interaction protein-DNA. We used the gel shift assay in order to study our proteins for the respectively targeted DNA amplicons.

**Fig 4. Activity test of TAL2 in the presence/absence of the DNA amplify**
Lane A is a negative control containing only the genetic material. The other lanes are an increase of the quantity of our proteins in contact with the same quantity of DNA amplify. TAL2 were designed to target the rpoB gene that are amplify PSR using two specific primers, which is a gene that’s present in Tuberculosis. Lane B and C contains 10 to 20 times more of proteins TAL2 than DNA amplify, there isn’t no shift visible. Lane D of rpoB there is a partial gel shift with a ratio of 1:30, meaning that there were serval TAL2 that was able to fixate itself of to the DNA amplify.

![T--Aix-Marseille--TAL2 gel shift 2.png picture.png](/wiki/images/1/1a/T--Aix-Marseille--TAL2_gel_shift_2.png_picture.png)

### Conclusion and discussion

The protein TAL2 was design in order to recognize a specific sequence on the rpoB gene in M.Tubercolosis (5’ CCGCTG 3’). This engineered TAL consist of repeat domain has a primary structure with two amino acids that differs in each domain, those two amino acids are a code for one nucleotide of the binding sequence selected. The SDS-PAGE (Fig 2) and Western Blot (Fig 3) show that our protein was well produced and purified, which allowed us to do an activity test.

The gel shift assay, shows the activity test of the different ratio of TAL2 in the presence of the same amount of DNA rpoB. There was a partial band shift (Fig.4D) present on the gel, which may be due to the insufficient quantity of protein TAL2 present. Thanks to the presence of this shift band allows to justify the fixation of TAL2 on rpoB gene with a ratio of 1:30, and the intensity of the shift shows approximately 30-40% of TAL2 were able to bind itself on to the gene.

### Perspectives

Two more test can be done. Firstly, the fixation of TAL2 needs improvements. By testing more ratio of TAL2 in the presence of rpoB gene. Secondly, the activity test needs to be done for the two other TALs (TAL1 and TAL3).

Antifreeze protein

Antifreeze proteins (AFPs) protects certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. We were able to produce and purified the Choristoneura fumiferana (Spruce budworm moth) (Archips fumiferana) AFP-Iu1 A family of expressed antifreeze protein genes from the moth, Choristoneura fumiferana.

### AFP-lu1 production

We were able to insert the AFP-lu1 gene into a plasmid containing the T7 promoter and RBS which is a psB1C3 vector. SDS-PAGE was performed and stained with Coomassie blue using cells containing this Biobrick, to verify the production of our protein we did different growing conditions to determine the best growing conditions by using two strains of E.coli bacteria :

* DH5α
* BL21 DE3

![T--Aix-Marseille--AF-lu production picture.png](/wiki/images/3/37/T--Aix-Marseille--AF-lu_production_picture.png)

**Figure 1. SDS-PAGE of the production in DH5a and BL21 DE3 of AF-lu1,** well 1 and 2 are induced at 30°C with 0.1M IPTG for 3hours in DH5a and BL21 DE3, respectively. Well 5 and 6 are not induced of AF-lu1 in DH5a and BL21 DE3 respectively. Well 3 and 4 are induced at 16°C with 0.1M IPTG overnight in DH5a and BL21 DE3, respectively.

We used 2 different strains of E. coli Dh5 alpha, and a BL21 strain. There are bands at approximatively 27kDa, that correspond to AF-lu1 molecular weight that was engineered.
After analyzing these results, the induction condition at 0.1M IPTG at 30 ° C is chosen for the rest of the experiments. As we can see wither our protein are amplify overnight or for 3hours theirs the same quantity of protein that are produced, and wither our protein were produced in BL21 DE3 or DH5a theirs also the same amount of protein that are produced.
So, we choose to use the proteins that was produced overnight in BL21 DE3.

### AFP-lu1purification

After having realized the AFP-lu1 production tests, the purification was realized in order to obtain pure protein fractions. For this, the E. coli BL21 cells were lysates and then are purified. Purification of the protein was realized with a HisTag column which is a manual chromatography system for quick, easy ad small quantity purification. The fusion of the histine tag with AFP-lu1 allows to separate our protein from the other proteins.

![T--Aix-Marseille--AF-lu purification picture.png](/wiki/images/1/19/T--Aix-Marseille--AF-lu_purification_picture.png)

**Fig 2. SDS-PAGE of AFP-lu1 after purification with HisTag column.**LD= loading sample, NR= non-restraint, E1 to E7 correspond to the eluted fractions.

**Fig 3. Western Blot analysis of AFP-lu1 after purification with HisTag column.** (a) LD= loading sample, NR= non-restraint, E1 to E3 correspond to the eluted fractions. (b) from E4 to E7 correspond to the eluted fractions.

![T--Aix-Marseille--AF-lu Western blot picture.png](/wiki/images/3/35/T--Aix-Marseille--AF-lu_Western_blot_picture.png)

After SDS-PAGE visualization (fig. 2), the histidine tag allows to purify the AFP-lu1 from the total proteins. An intense band is observable approximately 27 kDa corresponding to AFP-lu1. However, the 27kDa band is not observable in the no-restrained, it means that our protein was will held back in the column. After purification, a pool of fractions E3 to E7 is made. The deposit on the SDS-PAGE of the no-retained and loading sample fraction allows to check if our protein has been retained on the column. There isn’t no band at 27kDa of our protein, so our protein has been retained correctly on the column. The fractions from E1 to E7 present a band at 27kDa, our protein was eluted. To confirm the elution corresponding to the AFP-lu1, a Western Blot of the fractions corresponds to the elution, flow-through, as well as the loaded sample was realized (Fig.3). In the Well LD and NI there isn’t no band at 27kDa, which confirms that our protein was well retained on the column and in the fraction E1 to E7 present a band. The presence of each band shows the release of our protein. The HisTag on our protein allows the fixation of the primary antibodies and the second antibodies are used to give the color of the band. Now that the protein is purified, an activity test was able to be realized but there was no more time to finish.

Methylases

We amplify of the NlaIII and HhaI, DNA sequence with the oligo OGC35/36. After the amplification and the DNA clean up of gene blocks fragments that we received by IDT. We amplified these DNA fragments with the primers containing the floating tail. Each fragment have been amplified with different specific oligo that we designed. The agarose gel electrophoresis 1%. We carry out a PCR DNA clean up in all PCR products that we obtained. A digestion of the Psb1c3 have been made with de EcoRI and PstI restriction enzymes, and have been used to performed a SLIC with these NlaIII-Linker/CDB-Linker and HhaI-Linker/ CDB-Linker. After doing a bacterial transformation, we obtained colonies on which we realized PCR colony. The results are shown below

[![T--Aix-Marseille--Methylase PCR amplification picture.png](/wiki/images/e/e6/T--Aix-Marseille--Methylase_PCR_amplification_picture.png)](/File%3AT--Aix-Marseille--Methylase_PCR_amplification_picture.png)

Figure 1 : We see two bands that seem higher than when we have only methylases, which tells us that methylases are well linked with CBD. We can see a slight shift of methylases towards higher molecular weight, which accounts for the binding between the methylases and the cbd.

[![T--Aix-Marseille--Methylase PCR colo picture.png](/wiki/images/2/27/T--Aix-Marseille--Methylase_PCR_colo_picture.png)](/File%3AT--Aix-Marseille--Methylase_PCR_colo_picture.png)

Figure 2 : this Slic means good. We see that the molecular weight is good and it is a succession of methylase, in the well 1 we have the molecular weight scale then the well 2 is the NlaIII-M methylase and the well 3 is HhaI-M. then, then, between the two methylases, some wells work more than others.

TolA3/P3-N1-chromoprotein

The prefix and the suffix oligos have been designed to carry out PCR amplification. These oligos are suitable for all the proteins constructions of **TolA3-CBD** and the **Green chromoprotein-P3-N1**.
The 3’end of **Cellulose binding domain** (called CBD) primer was chosen in order to amplifiy the protein sequence by adding a binder at the 3’ end. To bind both proteins, a binder was placed on the oligo 5’ of **TolA3** DNA fragment. This oligo is complementary of the binder presented on the 3’ part of the chromoprotein oligo. This makes it possible to create a chimeric **TolA3** protein coupled to the **CBD**.
The 3’end of **Green chromoprotein** primer was chosen in order to amplifiy the protein sequence by adding a binder at the 3’ end. To bind both proteins, a binder was placed on the oligo 5’ of g3p DNA fragment (called P3-N1 protein). This oligo is complementary of the binder presented on the 3’ part of the chromoprotein oligo. This makes it possible to create a chimeric **P3-N1** protein coupled to the **Green chromoprotein**.

We amplify of the P3-N1, TolA3, CBD, and Green chomoprotein DNA sequence with the oligo OGC35/36. After the amplification and the DNA clean up of gene blocks fragments that we received by IDT. We amplified these DNA fragments with the primers containing the floating tail.. Each fragment heve been amplified with different specific oligo that we designed. The agarose gel electrophoresis 1% is shown below.

![T--Aix-Marseille--TolA3 P3-N1 agarose gel electrophoresis profil picture.jpg](/wiki/images/a/ab/T--Aix-Marseille--TolA3_P3-N1_agarose_gel_electrophoresis_profil_picture.jpg)

**Figure 1 : Agarose gel electrophoresis of the PCR products**

* Well1 : Size marker (smart Ladder)
* Well2 : TolA3 DNA fragment with the linker (floating tail)
* Well3 :P3-N1 DNA fragment with the linker (floating tail)
* Well4 : CBD DNA fragment with the linker (floating tail)
* Well5 : Green chromoprotein DNA fragment with the linker (floating tail)

We can see that all DNA fragments have been amplified, suggesting that the most of DNA fragments have received the binder.

We carry out a PCR DNA clean up in all PCR products. A digestion of the Psb1c3 have been made with de EcoRI and PstI restriction enzymes, and have been used to performed a SLIC with these TolA3-Linker/CDB-Linker and P3-N1-Linker/Green chromoprotein-Linker. After doing a bacterial transformation, we obtained colonies on which we realized PCR colony. The results are shown below

![T--Aix-Marseille--TolA3 P3-N1 PCR colony picture.png](/wiki/images/e/ec/T--Aix-Marseille--TolA3_P3-N1_PCR_colony_picture.png)

**Figure 2 : Agarose gel electrophoresis of the PCR colonies products**

* Well1 : Size marker (DNA Ladder)
* Well2 to 13: TolA3-CBD DNA fragment
* Well 14 to 25 : Green chromoprotein-P3-N1 DNA fragment

The TolA3-CBD construct should theoretically contain 783 base pairs. The results of the electrophoresis gel show Tol-CBD constructs around 800 bp while the P3-N1-chromoprotein constructs have sizes of about 600 base pairs.
The sequencing results showed that the P3-N1-Green chromoprotein constructs did not exhibit any protein sequence encoding the fusion protein. Only the presence of mRFP was demonstrated during sequencing.
The results of the sequencing, however, showed the presence of CBD in the Psb1C3 vector from the TolA3-CBD clones. Multiple alignment verification (MUSCLE) confirmed the presence of the coding sequence for the TolA3-CBD fusion protein in plasmid pSB1C3.

### Digestion verification by agarose gel electrophoresis

We’ve digested 3 plasmides of the clones TolA3-CBD with two differentes restriction enzyme such as EcoR-HFI and PstI-HF .We carry out three differents digestions for each sample, in order to verifyy the persence of the insert in the plasmid and to test the restrictions enzymes activities.
The digestion have been carry out for 1.30 hour at 37°C.

![T--Aix-Marseille--TolA3-CBD digestion verification picture.png](/wiki/images/d/de/T--Aix-Marseille--TolA3-CBD_digestion_verification_picture.png)

**Figure 3 : Digestion verification of the TolA3-CBD clones by agarose gel electrophoresis**

* Well 1 : Size marker (smart Ladder)
* Well 2, 6 and 10: pSB1C3 with TolA3-CBD insert which have been digested by EcoRI-HF.
* Well 3, 7 and 11: pSB1C3 with TolA3-CBD insert which have been digested by PstI-HF.
* Well 4, 8 and 12: pSB1C3 with TolA3-CBD insert which have been disgested by both restrictions enzymes (EcoRI-HF and PPstI-HF).

The results showed that all the clones tested contained the ToLA3-CBD insert within the Psb1c3 plasmid. The DNA fragments obtained around 2000 base pairs correspond to the linear psb1c3 plasmid whereas the bands located around 800 base pairs correspond to the TolA3-CBD insert.

### P3-N1/chromoprotein constructions

We tryed to performed an other SLIC in order to get the P3-N1 /Green chromoprotein construction. After doing a bacterial transformation, we obtained colonies on which we realized PCR colony. The results are shown below

![T--Aix-Marseille--P3-N1 PCR colony picture.png](/wiki/images/e/e2/T--Aix-Marseille--P3-N1_PCR_colony_picture.png)

**Figure 4 : Agarose gel electrophoresis of the PCR colonies products**

* Well1 : Size marker (DNA Ladder)
* Well2 to 8: Green chromoprotein-P3-N1 DNA fragment

The Green chromoprotein-P3-N1 construct should theoretically contain 1041 base pairs. The results of the electrophoresis gel show that the Green chromoprotein/ P3-N1 is around 1000 base pairs.
The results of the sequencing showed the presence of Green chromoprotein in the Psb1C3 vector from the Green chromoprotein/ P3-N1 clones. Multiple alignment verification (MUSCLE) confirmed the presence of the coding sequence for the Green chromoprotein/ P3-N1 fusion protein in plasmid pSB1C3.

Protocols

Competent cells preparation (for *E. coli K-12 DH5-alpha*)

1. The day before the preparation, make overnight starters with an isolated colony on an agar plate in 5 mL of LB in a 50 mL Erlenmeyer.
2. Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and pipette tips at -80°C for future use.
3. The next day, recover your starters and seed 1/100 of bacterial cells in 200mL of LB, then incubate at 37°C and 180rpm.
4. Keep measuring the OD600. When it’s between 0.4 and 0.6, recover your cells and pool them in cold 50 mL Falcon tubes.
5. Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
6. Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
7. Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
8. Take 100 μL aliquots in Eppendorf tubes and stock them at -80 °C.

Buffer 1 Preparation

| Product | For 40 mL |
| KAc (1M) | 1.2mL |
| MnCl2 (0.5M) | 4 mL |
| KCl (1M) | 4 mL |
| CaCl2 (0.1%) | 4 mL |
| Glycerol (80%) | 7.5 mL |
| H2O | 19.3 mL |

Buffer 2 Preparation

| Product | For 8,5 mL |
| KCl (1M) | 80µL |
| MOPS (1M) | 400 µL |
| CaCL2 (0,1M) | 6mL |
| Glycerol (80%) | 1,5 mL |
| H2O | 500 µL |

DNA transformation

1. Recover the right amount of competent cells tubes An tube serves as a negative control.
2. Recover 50-100µL of competent cells.
3. Add DNA (1-2µL) into the competent cells.
4. Incubate on ice for 20 minutes.
5. Incubate tube at 42°C for 1 minute for thermal choc.
6. Leave tubes on ice for 5 minutes.
7. Add 900 µL of LB (Lysogeny broth) per tube, then incubate for 1 hour at 37°C and 850rpm.
8. Centrifuge tube for 5 minutes at 4000rpm at RT. Get rid of 900µL of supernatant.
9. Resuspend and spread cells on Petri dishes with the desired antibiotic and incubate overnight at 37°C.

DNA digestion

For 250 ng of DNA

| Product | For 30 µL |
| DNA | depend on the DNA concentration |
| 10X buffer | 3 µL |
| Restriction enzyme 1 | 0.3 µL |
| Restriction enzyme 2 | 0.3 µL |
| MiliQ water | To 30 µL |

Incubate 3 hours at 37°C
Do a DNA clean up (protocole à ajouter)

DNA ligation

For 50ng of vector

| Product | For 20 µL |
| Vector | depend on the vector concentration |
| Insert | use NEBcalculator (ratio 3:1) |
| T4 10X buffer | 2 µL |
| T4 DNA ligase | 1 µL |
| MiliQ water | To 20 µL |

Incubate 2 hours at room temperature (or overnight at 16°C)

Polymerase spiral reaction (PSR)

|  |  |  |
| --- | --- | --- |
| **Components** | **For 25 uL** | **Final concentration** |
| **Bst DNA polymerase** | 1 ul | 320 U/ml |
| **dNTPs (10mM)** | 3,5 ul | 1.4 mM each |
| **Thermo Pol buffer 10X** | 2,5 ul | 1X (contains 2 MgSO4) |
| **Forward Primer** | 1 ul | 1.6 uM |
| **Reverse Primer** | 1 ul | 1.6 uM |
| **MgSO4** | 1,5 ul | 6mM (8mM total) |
| **DNA sample** | Variable (1ul) | >10 copies |
| **Betain (10 M)** | 1,5 ul | 0,6 M |
| **Nuclease free water** | To 25 ul |  |
| **Total Reaction Volume** | 25 ul |  |

Monarch PCR and DNA cleanup kit

All centrifugation steps are done at ~13000 rpm in a typical microcentrifuge. This ensures that all traces of buffer are eluted at each step.

1. A starting sample volume of 50 μL is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. Add 100 μL DNA Cleanup Binding Buffer to the 50 μL sample.

2. Insert a column into a collection tube, load sample onto the column and close the cap. Spin for 1 minute, then discard flow-through.

3. Re-insert column into the collection tube. Add 200 μL DNA Wash Buffer and spin for 1 minute. Discard flow-through.

4. Repeat Step 3. This step is recommended for removal of enzymes that may interfere with downstream applications.

5. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.

6. Add ≥ 15 μL of hot water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute the DNA.

7. Repeat step 6.

Monarch miniprep kit

All centrifugation steps are done at ~13000 rpm in a typical microcentrifuge. If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve. Store Plasmid Neutralization Buffer (B3) at 4°C after opening.

1. Pellet 1–5 mL bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.

2. Resuspend pellet in 200 μL Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.

3. Lyse cells by adding 200 μL Plasmid Lysis Buffer (B2) (blue/green). Invert the tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 5 minutes.

4. Neutralize the lysate by adding 400 μL of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until the color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.

5. Clarify the lysate by spinning for 10 minutes at ~13000pm.

6. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.

7. Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein, and endotoxin. Centrifuge for 1 minute. Discard flow-through.

8. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

9. Centrifuge for 1 minute one more time.

10. Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.

11. Add ≥ 15 μL Hot Water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

12. Repeat step 11.

IPTG induced protein production

## Production

1. Prepare 6 starters (20 mL LB and chloramphenicol): control BL21 (No plasmid), BL21 with the plasmid in duplicates, control DH5α (No plasmid), and DH5α with the plasmid in duplicates. Incubate overnight at 37°C and 180 rpm.

2. The next day, recover the overnight starters and measure the OD600.

3. Make 1/60 dilutions and incubate at 37°C to reach 0.7 in OD600.

4. Add (For one of the duplicates) IPTG (1M) (10µL for 20 mL) to induce the protein production in BL21 and DH5α with the plasmid.

5. Incubate at 16°C, 30°C, and 37°C for 5 hours.

6. Leave the remaining cultures at 37°C for 5 hours. Incubate the cultures at 16°C overnight.

7. After 5 hours, measure the OD600 and take 1 mL aliquots.

8. Centrifuge aliquots for 10 minutes at 13000 g. Discard supernatant and stock samples at -20°C.

9. For the remaining cultures, pour the remaining volumes into 50 mL Falcon tubes and centrifuge for 20 minutes at 4700 g and 4°C. Discard supernatant and stock samples at -20°C.

10. The next day, measure the OD600 of the culture at 16°C, take 1 mL aliquots, then repeat steps 8 and 9.

## Electrophoresis gel migration

1. Add TSTD (1X) at 50 µL/OD unit to the 1 mL aliquots. Do not resuspend.

2. Put samples in the dry bath incubator for 20 minutes

3. Prepare the migration apparel and charge the gel (20 µL for samples, and 5 µL for the ladders).

4. Start migration at 0.04 A for an hour.

5. Incubate gels in Coomassie blue for an hour.

6. Wash gels with acetic acid till they're transparent.

7. Once the gel is clear, add ddH2O and stock at 4°C.

Protein purification

## Cell lysis and protein extraction

1. Prepare a 1L starter cell cultures and incubate at the optimal conditions tested out during the first protein production.

2. Centrifuge for 20 minutes at 4500 rpm and 4°C . Discard supernatant.

3. Resuspend pellet in a lysis buffer (50 mM Tris-HCl (pH=8), 50 mM NaCl, 1 mM EDTA, 0.1 mg/L DNase, and an anti-protease tablet).

4. Put on agitator for 30 minutes at 4°C .

5. Disrupt the cells using the emulsiflex.

6. Ultracentrifuge for 35 minutes at 20000 rpm and 4°C .

7. Transfert supernatant into 50 mL Falcon tubes and discard pellets.

## His-tag Protein purification

1. The purification uses the [ÄKTA pure system](https://www.gelifesciences.com/en/bg/shop/chromatography/chromatography-systems/akta-pure-p-05844) with [HisTrap™](https://www.gelifesciences.com/en/de/shop/chromatography/resins/affinity-tagged-protein/histrap-hp-histidine-tagged-protein-purification-columns-p-00250) columns.

2. Wash the column with 5 H2O volumes to eliminate all ethanol.

3. Wash the column with 5 buffer A (50 Mm Tris-HCl (pH=8), 50 mM NaCl) volumes.

4. Inject the supernatant from the protein extraction in the HisTrap™ column. Discard flowthrough.

5. Elute the column with 10 buffer A volumes. Discard flowthrough.

6. Using a 0 to 100% gradient, elute the column with buffer B (Buffer A + 500 mM imidazol). Collect flowthrough gradually in 1 mL fractions.

7. Recover the collected fractions and verify the purification on a SDS-PAGE gel.

8. Quantify the purified protein on the NanoDrop.

## Protein purification by ion exchange column

The purification uses the [ÄKTA pure system](https://www.gelifesciences.com/en/bg/shop/chromatography/chromatography-systems/akta-pure-p-05844) system with [anion exchange columns](https://www.gelifesciences.com/en/si/shop/chromatography/prepacked-columns/ion-exchange/mono-q-anion-exchange-chromatography-column-p-00608#/overview).

1. Wash the column with 5 H2O volumes to eliminate all ethanol.

2. Wash the column with 5 buffer A (50 Mm Tris-HCl (pH=8), 50 mM NaCl) volumes.

3. Inject the supernatant from the protein extraction in the column. Collect flowthrough.

4. Elute the column with 10 buffer A volumes. Collect flowthrough.

5. Using a 0 to 100% gradient, elute the column with buffer B (50 Mm Tris-HCl (pH=8), 1 M NaCl). Collect flowthrough gradually in 1 mL fractions.

6. Recover the collected fractions and verify the purification on a SDS-PAGE gel.

7. Quantify the purified protein on the NanoDrop.

Western blot

Preparing a SDS-PAGE gel (4 gels recipe)

The SDS-PAGE gel has two main composites: a stacking gel and a separation gel. The following table shows the recipe to prepare them.
When putting them in the plaques pool the separation gel before the stacking gel, and leave to dry for 15 minutes.

| Product | Stacking | Separating gel |
| --- | --- | --- |
| Acrylamid | 600 µL | 6.25µL |
| Tris-HCl (3M, pH=8.8) | 2.64 mL | - |
| Tris-HCl (1M, pH=6) | Tris-HCl (1M, pH=6) | 600 µL |
| SDS (10%) | 50 µL | 200 µL |
| APS (10%) | 60 µL | 80 µL |
| TEMED | 5 µL | 10 µL |

When the gel is ready, prepare your samples (Dilute them in TSTD 1X at 50 µL/OD unit), then incubate for 10 minutes at 95°C.
Load the gel and run the migration system for 2 hours (10 minutes at 90V and 110 minutes at 120V).

### Western blot

1. Setup the migration sandwich: Whatman paper (+), nitrocellulose membrane, gel, Whatman paper (-). Soak in blotting buffer.

2. Put the assembly in the blotting system and run it at 250 mA and 1 minute/kDa.

3. Recover the membrane. Incubate overnight at 4°C or for an hour at RT on agitator (50 opm) in saturation buffer (TBS 1X, Tween20 0.1%, and milk 10%).

4. Wash membrane in washing buffer (TBS 1X and Tween20 0.1%) for 5 minutes at 50 opm. Repeat step two more times.

5. Dilute the primary antibiodies (1/1000) in TBS 1X, Tween20 0.1%, and milk 10%. Incubate membrane overnight at 4°C or an hour at RT and 20 opm.

6. Wash membrane in washing buffer (TBS 1X and Tween20 0.1%) for 5 minutes at 50 opm. Repeat step two more times.

7. Dilute the secondary antibiodies (1/5000) in TBS 1X, Tween20 0.1%, and milk 10%. Incubate membrane overnight at 4°C or for an hour at RT and 20 opm.

8. Wash membrane in washing buffer (TBS 1X and Tween20 0.1%) for 5 minutes at 50 opm. Repeat step two more times.

Sequence and Ligation-Independent Cloning (SLIC)

### Procedures

1. Digest vector with restriction enzyme(s) overnight, and purify the inearized vector with commercial PCR purification kit. Elute the DNA with elution buffer or 10 mM TrsCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the vector.

2. Amplify your gene of interest by PCR using primers with ≥ 15 mer homolog extension to the linearized vector end. We usally use 15 bp homlogy for single fragment cloning, and 20 bp homology for multiple fragment cloning. Purify the linearized vector with a commerciale PCR purification kit. Elute the DNA with elution buffer or 10 mM TrisCl, pH 8.0-8.5. Do not elute the DNA with watrer or TE. Measure the concentration of the insert(s).

3. Mix thelnearized vector and insert at a molar ratio of 1:2 in a 1:5 mL tube. An example is shown as follows. (Vector to insert molar ratio of : 1:1; to 1:7 works well but we asually use 1:2 for the single fragment cloning, 1:2:2 for multiple fragments cloning. An example of 3 fragment cloning is shown below with vector: insert 1: insert 2 molar ratio is : 1:2:2 as shown.

|  | Stock concentration | Volume added | Final concentration |
| --- | --- | --- | --- |
| Linearized vectoe (eg,5kb) | 100 ng/µL | 1 µL | 10 ng/µL |
| Insert1 (PCR product, eg, 1kg) | 40 ng/µL | 1µL | 4 ng/µL |
|  | 40 ng/µL | 1µL | 4 ng/µL |
| 10X BSA |  | 1µL | 1X |
| 10X NEB Buffer 2 |  | 1µL | 1X |
| H2O |  | Up to 10µL |  |

4. Add 0.5 µL of the T4 DNA polymerase (3 U/µL, NEB) to the mixture and incubate at room temperature for 2.5 min. We found that 0,5 (1,5 U ) of the T4 DNA polymerase gives the best result, but 0.2 µL gives more than sufficient number of colonies.

5. Put the reaction mixture on ice immmediately to stop the reaction and incubate on ice for 10 min.

6. Thaw chamivcally competent E.coli cells on ice for 10 minutes

7. For single fragment clonning, gently mix the cells with 10 µl of the reaction mixture and incubate the cells on ice for 20 min. For multiple fragment cloning, mix the cells with 3-5 µL of the reactants.

8. Incubate he cells on ice for 30 minutes.

9. Heat shock the cells at 40°c for 45 secondes.

10. Incubate the cells on ice for 3 minutes.

11. Add 900 (or 950) µL of LB broth to 100 (or 50) µL of the cells.

12. Incubate the cells at 37°C for 1 hour.

13. Plate the cells on agar plates containing suitable antibiotics (eg, 100 µg/ml amplicillin). We usually spread 10 to 20 µL of the cells onto an agar to get optimal number of colonies per plate for single fragment cloning, and 100 µL per plate for multiple fragments cloning. In case of 10-20 µL spreading, cells needs to be further diluted with 80-90 µL LB broth for suitable spread spreading.

14. Incubate the plates at 37°C for 16 hours and analyze the colonies.

Gel shift

Reactional buffer (conserved at -20°C)

|  | Stock | 5X | Volume (for 1mL) |
| --- | --- | --- | --- |
| Tris-HCl (pH 7,2) | 1M | 125 mM | 125 µL |
| MgCl2 | 1M | 50mM | 50 µL |
| CaCl2 | 0,1M | 5mM | 50 µL |
| EDTA (pH 8) | 0,5M | 2,5mM | 5 µL |
| KCl | 1M | 250mM | 250 µL |
| Glycerol | 80% | 25% | 300 µL |
| Water |  |  | 220 µL |

Acrylamide gel: 10% (29:1) without SDS and without stacking

For 10mL:

* 5,35mL of water
* 3,3mL acrylamide (29:1; 30%)
* 1,25mL Tris pH 8,8 3M
* 100 µL APS 10%
* 6 µL Temed

To be prepared the day before

* Make a pre-run during one hour in TAE 1X at RT and 80V constant

Preparation of the reactional mix (to prepare during the pre-run)
DNA: between 50 and 100ng (around 20 nM) : 1µL (to be adjusted slowly)
Protein: adjusted depending on ratio Prot/DNA
Buffer 5X: 4µL
H20: qsp 20

**Incubation of samples** during 20-30 minutes at 20°C without agitation in thermomix, then whole samples are loaded in the gel
**Migration** during two hours in TAE 1X at RT and 80V
**Coloration** during 10 min in red gel.

| Ratio Protein:DNA | 0 | 10 | 20 | 30 | 40 |
| --- | --- | --- | --- | --- | --- |
| DNA | 1 µL | 1 µL | 1 µL | 1 µL | 1 µL |
| Protein | 0 µL | 1 µL | 2 µL | 3 µL | 4 µL |
| Buffer 5X | 4 µL | 4 µL | 4 µL | 4 µL | 4 µL |
| Water until 20 µL | 15 µL | 14 µL | 13 µL | 12 µL | 11 µL |

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Aix-Marseille/Results

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[HOME](https://2019.igem.org/Team%3AAix-Marseille)

[TEAM](https://2019.igem.org/Team%3AAix-Marseille/Team)

PROJECT

[Description](https://2019.igem.org/Team%3AAix-Marseille/Description)
[Design](https://2019.igem.org/Team%3AAix-Marseille/Design)
[Experiments](https://2019.igem.org/Team%3AAix-Marseille/Experiments)
[Notebook](https://2019.igem.org/Team%3AAix-Marseille/Notebook)
[Contribution](https://2019.igem.org/Team%3AAix-Marseille/Contribution)
[Results](https://2019.igem.org/Team%3AAix-Marseille/Results)
[Model](https://2019.igem.org/Team%3AAix-Marseille/Model)

PARTS

[Parts Overview](https://2019.igem.org/Team%3AAix-Marseille/Parts)
[Basic Parts](https://2019.igem.org/Team%3AAix-Marseille/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AAix-Marseille/Composite_Part)

HUMAN PRACTICES

[Integrated Human Practices](https://2019.igem.org/Team%3AAix-Marseille/Human_Practices)
[Education & Engagement](https://2019.igem.org/Team%3AAix-Marseille/Public_Engagement)
[Collaborations](https://2019.igem.org/Team%3AAix-Marseille/Collaborations)
[News](https://2019.igem.org/Team%3AAix-Marseille/News)

[SAFETY](https://2019.igem.org/Team%3AAix-Marseille/Safety)

ACKNOWLEDGEMENTS

[Attributions](https://2019.igem.org/Team%3AAix-Marseille/Attributions)
[Sponsors](https://2019.igem.org/Team%3AAix-Marseille/Sponsors)

[MEDAL CRITERIA](https://2019.igem.org/Team%3AAix-Marseille/Medal_criteria)
[JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Aix-Marseille)

Results

After 6 months, we were able to realize this project with all the hard work we put in. We were able to optimize an existent chromoprotein which allowed us to reach a bronze criterion. We were also able to produce a functional TAL, that’s able to recognize a specific sequence, which was able to answer the silver medal criterion. We were able to meet the requirements for a gold medal criterion with a successful model, the modeled was able to bring forth the rate of mortality by ***M.Tuberculosis*** and the survival rate using our detection kit.

Furthermore, we were able to further our project thanks to the integrated human practice by public engagement. That allowed us to optimize our kit and adapt it to the public needs. Those exchanges allowed our project to meet a silver criterion and the integration of the human practice meaning adapting our project to the answer of the surveys, permitted us to reach a Golden criterion.

Wetlab

### PSR

We used the [PSR (polymerase spiral reaction)](/Team%3AAix-Marseille/Design#anchor_psr "Team:Aix-Marseille/Design") method because it meets the criteria of our test. Indeed, it is a fast method, inexpensive and doesn’t require a great technical and material capacity. The important step of this achievement was to design specific pairs of primers that can amplify the DNA with a high specificity, but also sensitive to low concentrations of DNA.

Once the PSR method was well controlled, we were able to test our primers on a wide variety of bacterial strains and concluded that our primers had high [specificity](/Team%3AAix-Marseille/Experiments "Team:Aix-Marseille/Experiments"), which is essential for the diagnosis of ***M.Tuberculosis***.

Sensitivity of this method is another important criterion, to be able to diagnose patients in an early stage of infection that would be undetectable by a microscope for example. After testing the sensitivity of this method with different DNA concentration, we noticed that our primers have a sensitivity equivalent to a PCR test. We can see 10X more sensitive for ***M.Smegmatis***, and the limit of detection is up to 100 copies of genomic DNA per microliter, which is a good limit of detection for a testing based in molecular biology. [Click here for more](/Team%3AAix-Marseille/Experiments "Team:Aix-Marseille/Experiments").

### TAL

[TALs](/Team%3AAix-Marseille/Design#anchor_tal "Team:Aix-Marseille/Design") proteins are naturally produced in genus ***Xanthomonas*** bacteria that are able to infect plants by binding itself specifically to the promoter. In our project, we designed our TALs protein to recognize a sequence of choice. We were able to produce it in ***E. coli***, we successfully produced, and purified a functional TAL2 ([BBa\_K2915275](http://parts.igem.org/Part%3ABBa_K2915275)). Then tested it on a [gel shift](/Team%3AAix-Marseille/Experiments "Team:Aix-Marseille/Experiments"), which confirms that our TAL2 is functional and specific to a specific sequence chosen.

We’re able to choose our sequence of choice by basing on the DNA fragment amplify (5’ CCGCTG 3’). The sequence that was chosen are found on rpoB gene and katG gene of ***M. Tuberculosis***, dependent on which primers are used to amplify the DNA. There’s a beginning of sensitivity detected in between the DNA amplify and TAL2 with a ratio of 1:30 ([see more](/Team%3AAix-Marseille/Experiments "Team:Aix-Marseille/Experiments")). This part [BBa\_K2915275](http://parts.igem.org/Part%3ABBa_K2915275) is our validated part for a Silver medal criteria.

Biobrick characterization

We successfully characterized an existing iGEM biobrick ([BBa\_K1033929](http://parts.igem.org/Part%3ABBa_K1033929)). We determined its extinction coefficient and its thermal stability. [See here for more details](/Team%3AAix-Marseille/Contribution "Team:Aix-Marseille/Contribution")

Model

To help shape our [design](/Team%3AAix-Marseille/Design "Team:Aix-Marseille/Design"), we created an [epidemiological model](/Team%3AAix-Marseille/Model "Team:Aix-Marseille/Model") based on [***Abu-Raddad et al's***](https://doi.org/10.1073/pnas.0901720106) paper. The model evaluates different cases of Tuberculosis dissemination and evolution throughout many years. Using the model, we saw by which disease state the population’s death rate is mostly affected. Then, we could concentrate our efforts on the diagnosis of that state. The increase in diagnosis efficiency showed promising results. Simulations showed that our diagnosis procedure helped decrease TB cases. The model is robust because it uses data from the world health organization and a complete scientists consortium. Though the model is complex; it includes multiple parameters. It has already allowed us to draw several conclusions. As the model improves and the parameters are refined, other conclusions can follow. Thus, our model helps understand critical aspects of the suggested design.

[![T--Aix-Marseille--Modelling All z barplot.png](/wiki/images/9/9f/T--Aix-Marseille--Modelling_All_z_barplot.png)](/Team%3AAix-Marseille/Model "Team:Aix-Marseille/Model")

Integrated Human Practices

The [engagement](/Team%3AAix-Marseille/Public_Engagement "Team:Aix-Marseille/Public Engagement") with the public, the communication and the sharing of our project was very important for us, in order to make known the stakes of the tuberculosis and its diagnosis, but also to make known the synthetic biology and the iGEM. [Exchanges with specialists](/Team%3AAix-Marseille/Human_Practices "Team:Aix-Marseille/Human Practices")  (biologists and doctors) has been crucial for the development of our kit to make it easy to use, cheap, effective, accurate and accessible.
Our [Human Practices and Public Engagement](/Team%3AAix-Marseille/Human_Practices "Team:Aix-Marseille/Human Practices") were very strong, fulfilling the silver medal criteria, and we also integrated our work on human practices into the design of the project as gold medal criteria.