iGEM 2018 — Team UCLouvain

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UCLouvain/Notebook

[![](https://static.igem.org/mediawiki/2018/5/57/T--UCLouvain--logo-alpha.png)](https://2018.igem.org/Team%3AUCLouvain)* Our project

  [Context](https://2018.igem.org/Team%3AUCLouvain/Context)
  [Description](https://2018.igem.org/Team%3AUCLouvain/Description)
  [Design](https://2018.igem.org/Team%3AUCLouvain/Design)[Experiments](https://2018.igem.org/Team%3AUCLouvain/Experiments)
  [Results](https://2018.igem.org/Team%3AUCLouvain/Results)
  [Demonstrate](https://2018.igem.org/Team%3AUCLouvain/Demonstrate)
  [Perspectives](https://2018.igem.org/Team%3AUCLouvain/Improve)
* In the lab

  [Notebook](https://2018.igem.org/Team%3AUCLouvain/Notebook)
  [Attributions](https://2018.igem.org/Team%3AUCLouvain/Attributions)
  [InterLab](https://2018.igem.org/Team%3AUCLouvain/InterLab)
* Parts

  [Overview](https://2018.igem.org/Team%3AUCLouvain/Parts)
  [Basic parts](https://2018.igem.org/Team%3AUCLouvain/Basic_Part)
  [Composite parts](https://2018.igem.org/Team%3AUCLouvain/Composite_Part)
* [Safety](https://2018.igem.org/Team%3AUCLouvain/Safety)
* Human practices

  [Integrated Human Practices](https://2018.igem.org/Team%3AUCLouvain/Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3AUCLouvain/Public_Engagement)
* Awards

  [Best therapeutic project](https://2018.igem.org/Team%3AUCLouvain/awards/Best_therapeutic)

  [Entrepreneurship](https://2018.igem.org/Team%3AUCLouvain/Entrepreneurship)
  [Public Engagement](https://2018.igem.org/Team%3AUCLouvain/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AUCLouvain/Human_Practices)
* About us

  [Our Team](https://2018.igem.org/Team%3AUCLouvain/Team)
  [Our University](https://2018.igem.org/Team%3AUCLouvain/University)
  [iGEM Collaborations](https://2018.igem.org/Team%3AUCLouvain/Collaborations)
  [Our Sponsors](https://2018.igem.org/Team%3AUCLouvain/Sponsor)
  [Our Partners](https://2018.igem.org/Team%3AUCLouvain/Partners)
* [Judging form](https://igem.org/2018_Judging_Form?team=UCLouvain)

Notebook

## OUR LONG SUMMER!

#### [**JUNE 2018**](#collapse1)

#### [**JULY 2018**](#collapse2)

#### Week 1

* start of interlab’s experiments

#### Week 2

* design of our pBullet plasmid, capable of conjugation and recombination of a Crispr/Cas9 system, targeting RFP and AmpR gene, with a biosafety component (Lambda-Pir dependent)
* Interlab

#### Week 3

* Complete modification of our plasmid’s design
* Interlab

#### Week 4

* Culture and Minipreps of the pCas plasmid
* Enzyme digestion of Cas9 gene ( promoter and terminator too)
* Phenol/Chloroform purification of the Cas9 cassette
* Preparation of electrocompetent cells (S17-1) to receive our plasmid
* Order (at IDT) for our synthetic sgRNA part (targeting RFP and AmpR gene)
* Interlab

#### [**AUGUST 2018**](#collapse3)

#### Week 1

* Culture and phenol/Chlo purification of the plasmid pK18mob (our mobilizable plasmid, which will receive the Cas9 and sgRNA)
* Minipreps of the pK18mob plasmid
* PCR to amplify the Cas9 gene (with tracer part) of pCAS plasmid

#### Week 2

* Single/Double enzymatic digestion of pK18mob plasmid to check
* Minipreps, Proteinase K and Phenol/Chloroform purification after a PCR of Cas9
* Double enzymatic digestion of the Cas9 cassette
* Ligation of the Cas9 cassette into the pK18mob plasmid + verification of the result with enzymatic digestion and PCR
* Minipreps of the new plasmid (pK18mob+Cas9 cassette)
* Transformation of the new plasmid into the pUC19 strain
* PCR and purification of the Cas9 cassette (with the tracer part of the Cas9)
* Receipt of the IDT order (our synthetic sgRNA targeting RFP and AmpR)

#### Week 3

* Stability test of our receiver plasmid (low copy plasmid pSB4K5\_BBa\_J04450)
* Enzymatic digestion (linearization) of our biosafety plasmid (pSW23T carrying R6K Ori and Lambda-Pir dependent)
* Enzymatic digestion of the Cas9 cassette (tracer version) and ligation into pK18mob plasmid
* Verification of the sgRNA (received from IDT) by Miniprep and double enzymatic digestion
* Transformation of the sgRNA (with and without NheI site) (note: 3 of the 6 orders received from IDT was empty !)
* Transformation of the new pK18mob into chemically competent cells
* Glycerol stock and Minipreps of the sgRNA
* Glycerol stock and Minipreps of the pK18mob with Cas9 cassette (tracer version)
* Verification by enzymatic digestion of the plasmid insertion

#### Week 4

* Double enzymatic digestion of the sgRNA targeting RFP and the new pK18mob (with Cas9 cassette)
* Ligation of the sgRNA targeting RFP with the new pK18mob (with Cas9 cassette)
* First test to check the conjugation protocols
* Minipreps and gel verification of the sgRNA targeting RFP and the new pK18mob (with Cas9 cassette)
* Sequencing of the pK18mob + Cas9 cassette and sgRNA (with and without NheI)
* Transformation of the new plasmid (pK18mob + Cas9 cassette + sgRNA targeting RFP)
* Verification of all our strain with antibiograms
* PCR modification, minipreps and gel verification of the plasmid pSW23T to modify the MCS

#### [**SEPTEMBER 2018**](#collapse4)

#### Week 1

* Sequencing of the pK18mob + Cas9 cassette (tracer version)
* Concentration verification (Nanodrop) of our killer plasmid (targeting RFP gene)
* Transformation into S17-1 of our killer plasmid (targeting RFP gene)
* Verification of efficiency (PCR diagnostic, enzymatic digestion, gel) of the BaeI enzyme (for our new biobrick system)
* PCR diagnostic of our killer plasmid (two version of the Cas9 with and without tracer)
* New clonage approach test for the sgRNA (NheI version) with pGem T-easy
* Verification test of the efficiency of the Cas9 and sgRNA in pK18mob plasmid + stability test of our killer plasmid with our reporter plasmid.
* Electroporation and PCR of our killer plasmid in S17-1 Lambda-Pir
* PCR diagnostic of the pGem T easy
* Receipt of order from IDT of the sgRNA targeting AmpR gene.
* Enzymatic digestion and verification of the sgRNA targeting AmpR gene.
* Ligation and electroporation of our plasmid pK18mob with Cas9 cassette and the new sgRNA targeting AmpR gene.
* Interruption of all manipulation concerning both sgRNA (with and without NheI site) and the Cas9\_tracer\_version. (No DNA in the IDT order)

#### Week 2

* PCR verification after transformation of our new plasmid targeting AmpR gene + glycerol stock
* Minipreps and enzymatic digestion to check the presence of the sgRNA
* Verification of Crispr/Cas9 efficiency of our killer plasmid
* Enzymatic digestion, ligation, electroporation and verification of our biosafety plasmid (pSW23T targeting RFP and AmpR gene)
* Transformation + verification of our differents plasmids in E. coli strains.
* Sequencing of our killer plasmid targeting AmpR gene.
* First test of conjugation in a liquid phase
* Cleaning of the academic laboratory

#### Week 3

* Gibson + PCR amplification of pUC19
* Verification of the IDT order (our new biobrick)
* Transformation and PCR verification of the insertion of the Cas9+sgRNA cassette in the new pSW23T.
* Verification of the new IDT order (sgRNA with GFP reporter gene) : No DNA.
* Second verification of the efficiency of our Crispr/cas9 system
* Test of selection of our donor and receiver strain over NAC.
* Conjugation test 2 in liquid phase
* Conjugation test in plate with IPTG/X-Gal system
* Gel control of the last conjugation

#### Week 4

* Conjugation test of our donor strain (S17-1 with our killer plasmid targeting AmpR gene) and a E.coli strain carrying TEM1 gene in his chromosome.
* Enzymatic digestion and verification of a new reporter plasmid (with GFP AmpR and pSC101)
* Conjugation test with differents donor and receiver strains
* Verification of conjugation efficiency between donor strain and receiver strain, with IPTG/X-Gal system

#### [**OCTOBER 2018**](#collapse5)

#### Week 1

* preparation of strains for fluorescent microscopy
* Fluorescent microscopy assay
* Conjugation “in plate” test with differents donor and receiver strains

#### Week 2

* Quantitative conjugation “in plate” test with differents donor and receiver strains
* Conjugation “cross” test between our donor and receiver strains.
* Wiki writting

####

* Wiki writting and preparation of the poster
* Cleaning of the L2 laboratory


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UCLouvain/Experiments

[![](https://static.igem.org/mediawiki/2018/5/57/T--UCLouvain--logo-alpha.png)](https://2018.igem.org/Team%3AUCLouvain)* Our project

  [Context](https://2018.igem.org/Team%3AUCLouvain/Context)
  [Description](https://2018.igem.org/Team%3AUCLouvain/Description)
  [Design](https://2018.igem.org/Team%3AUCLouvain/Design)[Experiments](https://2018.igem.org/Team%3AUCLouvain/Experiments)
  [Results](https://2018.igem.org/Team%3AUCLouvain/Results)
  [Demonstrate](https://2018.igem.org/Team%3AUCLouvain/Demonstrate)
  [Perspectives](https://2018.igem.org/Team%3AUCLouvain/Improve)
* In the lab

  [Notebook](https://2018.igem.org/Team%3AUCLouvain/Notebook)
  [Attributions](https://2018.igem.org/Team%3AUCLouvain/Attributions)
  [InterLab](https://2018.igem.org/Team%3AUCLouvain/InterLab)
* Parts

  [Overview](https://2018.igem.org/Team%3AUCLouvain/Parts)
  [Basic parts](https://2018.igem.org/Team%3AUCLouvain/Basic_Part)
  [Composite parts](https://2018.igem.org/Team%3AUCLouvain/Composite_Part)
* [Safety](https://2018.igem.org/Team%3AUCLouvain/Safety)
* Human practices

  [Integrated Human Practices](https://2018.igem.org/Team%3AUCLouvain/Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3AUCLouvain/Public_Engagement)
* Awards

  [Best therapeutic project](https://2018.igem.org/Team%3AUCLouvain/awards/Best_therapeutic)

  [Entrepreneurship](https://2018.igem.org/Team%3AUCLouvain/Entrepreneurship)
  [Public Engagement](https://2018.igem.org/Team%3AUCLouvain/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AUCLouvain/Human_Practices)
* About us

  [Our Team](https://2018.igem.org/Team%3AUCLouvain/Team)
  [Our University](https://2018.igem.org/Team%3AUCLouvain/University)
  [iGEM Collaborations](https://2018.igem.org/Team%3AUCLouvain/Collaborations)
  [Our Sponsors](https://2018.igem.org/Team%3AUCLouvain/Sponsor)
  [Our Partners](https://2018.igem.org/Team%3AUCLouvain/Partners)
* [Judging form](https://igem.org/2018_Judging_Form?team=UCLouvain)

Experiments

### Miniprep

Miniprep were done with Sigma GenElute Plasmid Miniprep Kit
See protocol at <https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/pln70bul.pdf>

### Enzyme digestion protocols

Digestion were done with NEB (NewEngland BioLabs) enzymes, according to the official protocol
See <https://nebcloner.neb.com/#!/redigest>

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UCLouvain/Results

[![](https://static.igem.org/mediawiki/2018/5/57/T--UCLouvain--logo-alpha.png)](https://2018.igem.org/Team%3AUCLouvain)* Our project

  [Context](https://2018.igem.org/Team%3AUCLouvain/Context)
  [Description](https://2018.igem.org/Team%3AUCLouvain/Description)
  [Design](https://2018.igem.org/Team%3AUCLouvain/Design)[Experiments](https://2018.igem.org/Team%3AUCLouvain/Experiments)
  [Results](https://2018.igem.org/Team%3AUCLouvain/Results)
  [Demonstrate](https://2018.igem.org/Team%3AUCLouvain/Demonstrate)
  [Perspectives](https://2018.igem.org/Team%3AUCLouvain/Improve)
* In the lab

  [Notebook](https://2018.igem.org/Team%3AUCLouvain/Notebook)
  [Attributions](https://2018.igem.org/Team%3AUCLouvain/Attributions)
  [InterLab](https://2018.igem.org/Team%3AUCLouvain/InterLab)
* Parts

  [Overview](https://2018.igem.org/Team%3AUCLouvain/Parts)
  [Basic parts](https://2018.igem.org/Team%3AUCLouvain/Basic_Part)
  [Composite parts](https://2018.igem.org/Team%3AUCLouvain/Composite_Part)
* [Safety](https://2018.igem.org/Team%3AUCLouvain/Safety)
* Human practices

  [Integrated Human Practices](https://2018.igem.org/Team%3AUCLouvain/Human_Practices)
  [Education & Engagement](https://2018.igem.org/Team%3AUCLouvain/Public_Engagement)
* Awards

  [Best therapeutic project](https://2018.igem.org/Team%3AUCLouvain/awards/Best_therapeutic)

  [Entrepreneurship](https://2018.igem.org/Team%3AUCLouvain/Entrepreneurship)
  [Public Engagement](https://2018.igem.org/Team%3AUCLouvain/Public_Engagement)
  [Integrated Human Practices](https://2018.igem.org/Team%3AUCLouvain/Human_Practices)
* About us

  [Our Team](https://2018.igem.org/Team%3AUCLouvain/Team)
  [Our University](https://2018.igem.org/Team%3AUCLouvain/University)
  [iGEM Collaborations](https://2018.igem.org/Team%3AUCLouvain/Collaborations)
  [Our Sponsors](https://2018.igem.org/Team%3AUCLouvain/Sponsor)
  [Our Partners](https://2018.igem.org/Team%3AUCLouvain/Partners)
* [Judging form](https://igem.org/2018_Judging_Form?team=UCLouvain)

Results

## Reporter Plasmid Construction

![image](https://static.igem.org/mediawiki/2018/f/fc/T--UCLouvain--project-result-4.jpeg)

Figure 1: Recipient bacteria carrying our reporter plasmid (AmpR and RFP)

## Killer Plasmid : design and construction

### Design

We received a mobilizable plasmid (pK18mob) carrying Kanamycin resistance gene and OriT. Our purpose in to combine its conjugative capabilities with the Crispr/Cas9 system, so the final killer plasmid will be able to conjugate and to cut
a target gene (the RFP gene and Ampicillin resistance gene). So, from the pK18mob, we have inserted the Crispr/Cas9 system : the Cas9 gene, and a single guide RNA.

## Efficiency of conjugation.

### Test #1 conjugation efficiency: transfer of the mobilizable plasmid pK18mob

To verify the efficiency of transfer of the mobilizable plasmid pK18mob, we conjugated the recipient strain DH5ɑ containing the plasmid pSB4A5\_J04450 with the donor strain S17-1 containing the plasmid pK18mob (WT). The result of the
conjugation was spread on LB/Xgal/IPTG box without selection pressure. The counting of strains is based on the expressed phenotype. Donors include lactose operon and are blue in appearance, recipient cells that have not conjugated
express RFP. While the transfer of the plasmid pK18mob (WT) restores the lactose operon in DH5ɑ by transferring the intact LacZɑ gene. The simultaneous expression of the lactose operon and the rfp gene produces a purple phenotype with
DH5ɑ bacteria that have received the plasmid that can be mobilized by conjugation.

![image](https://static.igem.org/mediawiki/2018/f/fb/T--UCLouvain--project-result-3.jpeg)

Figure 5: Differentiation of strains based on the expressed phenotype.

From left to right, "red" colony: DH5a with pSB4A5\_J04450 (non-conjugated recipient), "blue" colony: S17-1 with pK18mob (donor) and "purple" colony: DH5a with pSB4A5\_J04450 and pK18mob (recipient having conjugated).

After 2 hours of conjugation on solid LB medium, the cells are collected and diluted in PBS 1X before being spread on LB/Xgal/IPTG plate per 100µl. Colony counts are carried out three times for each dilution.

![image](https://static.igem.org/mediawiki/2018/b/b9/T--UCLouvain--project-result-2.png)

Table 1: Count after conjugation (transfer of mobilizable plasmid pK18mob).

The ratio of donor (D) to recipient (R) is 0.172, i. e. D:R ≈ 1:6. The effectiveness of the conjugation is calculated by the ratio between the number of conjugated recipients (R+) divided by the total number of recipients (R+ and R-),
i.e. 61% effectiveness, of recipient bacteria having received the mobilizable plasmid.

## Efficiency of our Crispr/Cas9 system.

### Test #1 of our Crispr/Cas9 efficiency

To verify the efficiency of Crispr/Cas9 system of our plasmid, we have transformed the killer plasmid in a DH5ɑ strain carrying the RFP reporter plasmid (pSB4A5\_J04450). If our CRISPR/Cas9 plasmid system work well, it will cut the RFP gene
and the recipient bacteria (red and fluorescent) will appear white.

![image](https://static.igem.org/mediawiki/2018/5/5c/T--UCLouvain--project-result-5.jpeg)

Verification of our Crispr/Cas9 efficiency.

On the left : the recipient cells carrying the RFP reporter plasmid. On the right: the recipient cells carrying the RFP reporter plasmid and our Crispr/cas9 killer plasmid, targeting the RFP gene.

![image](https://static.igem.org/mediawiki/2018/c/c8/T--UCLouvain--project-result-15.png)

Table 2: Count after transformation by pK18mob\_CAS9 with or without sgRNA anti RFP.

The transformation of a donor bacterium by a plasmid containing Cas9 and its RNA anti RFP guide shows 100% suppression of the red phenotype in the transformed bacteria.

* As we can see, all of our transformed bacteria appears in red. Our Crispr/Cas9 system seems to have an efficiency of 100%

### Biomolecular confirmation of our Crispr/Cas9 system

An enzyme digestion (PstI) was performed on the same strains to check that the recipient plasmid was correctly cut after the transformation of our killer plasmid.

![image](https://static.igem.org/mediawiki/2018/c/c6/T--UCLouvain--project-result-8.jpeg)

Biomolecular confirmation of the effect of CRISPR/Cas9 on the reporter plasmid: miniprep on colony and linear digestion with PstI.

SL: SmartLadder, 1 to 3: killer plasmids: respectively, without sgRNA, with sgRNA anti-RFP and with sgRNA anti-TEM1, 4: reporter plasmid with TEM-1 and RFP in DH5ɑ, 5 to 7: transformation of DH5ɑ (with reporter plasmid) with the killer
plasmid without sgRNA, 8 to 9: transformation of DH5ɑ (with reporter plasmid) with the killer plasmid with sgRNA anti TEM-1, 10 to 12: transformation of DH5ɑ (with reporter plasmid) with the killer plasmid with sgRNA anti RFP.

* This plasmid digestion confirms that the reporter plasmid carrying the rfp and TEM-1 genes has been correctly cut by our Crispr/Cas9 system, both by the RNA guide targeting RFP and the one targeting the resistance gene.

### Tests of Conjugation

Different test were performed to verify the conjugation and/or Crispr/Cas9 system efficiency.

#### Conjugation “Cross” test

the purpose of this test is the verify the conjugation and the efficiency of our system by crossing, in a agar plate, a recipient strain (carrying our RFP reporter plasmid) with both our control donor strain (S17-1 with our plasmid without
sgRNA) and our donor strain (S17-1 with our killer plasmid with the sgRNA).

##### Cross test 1 with RFP Killer plasmid

Recipient strain: DH5ɑ strain (E. coli) with the RFP rapporter plasmid.
Donor strain : S17-1 strain with our RFP killer plasmid.
Control strain : S17-1 strain without our RFP sgRNA.

![image](https://static.igem.org/mediawiki/2018/4/4f/T--UCLouvain--project-result-14.jpeg)

Cross test 1.

A: Vertically, the recipient cell DH5ɑ (with RFP reporter plasmid pSB4A5\_J04450). Horizontally, up: the control donor strain S17-1 (pK18mob\_CAS9 without sgRNA), down: the donor strain S17-1 (pK18mob\_CAS9 with sgRNA anti-RFP cutting
reporter plasmid).

##### Cross test 2 with AmpR killer plasmid

Recipient cell: DS984 strain (E. coli CmR) with the RFP rapporter plasmid (AmpR).
Donor strain : S17-1 strain with our Amp killer plasmid.
Control strain : S17-1 strain without our AmpR sgRNA.

![image](https://static.igem.org/mediawiki/2018/4/4c/T--UCLouvain--project-result-1.jpeg)

![image](https://static.igem.org/mediawiki/2018/3/3d/T--UCLouvain--project-result-10.jpeg)

Cross test 2. Vertically : the recipient cell (with RFP reporter plasmid). Horizontally : Up: the donor strain (cutting AMP gene) Down: the control donor strain (without cutting Amp gene)

#### Conjugation “in plate” test

Before conjugation test, our strain (donor, recipient and control) was precultured O/N from glycerol stock.
The different strain used are:

* Donor : S17-1 with our killer plasmid (targeting RFP or AmpR)
* Control : S17-1 with our conjugative plasmid pK18mob, with Cas9 BUT without the sgRNA (no targeting)
* Recipient : Dh5alpha, or DS984 (with CmR gene in chromosome) or TOP10 (with a TEM1 gene in chromosome). Each of the recipient strain have our reporter plasmid (with RFP or GFP)

The pre cultured strain were centrifuged, and resuspended in fresh LB, and keeped at 37°C for about one hour the regenerate the pili. Note: for first tests we used PBS to wash antibiotics of precultured strain, but we don’t use it anymore,
to prevent bacteria from stress. Then, 5ul of each strain (donor, recipient and control) were spotted on a LB plate. For conjugation, 5ul of donor strain (and also control strain) were spotted over 5ul of the recipient strain. The plate
was incubated at 37°C between 3 hours and 20 hours. After than, each spot (donor, recipîent, control, two conjugative) was resuspended on liquid LB and some dilution was done (from 10-2 to 10-6). Each dilution was put on LB plate with
adequate antibiotics and putted at 37°C O/N, to check the result of conjugation and/or Crispr/Cas9 system.

![image](https://static.igem.org/mediawiki/2018/d/d7/T--UCLouvain--project-result-6.jpeg)

Classic conjugation assay used for our test

#### Conjugation in plate test 1 (non quantitative) targeting AmpR (TEM1)

* Donor (D) : S17-1 with our killer plasmid (sgRNA targeting AmpR, resistance: KanR)
* Control (CTRL) : S17-1 with our conjugative plasmid pK18mob, with Cas9 BUT without the sgRNA (no targeting). KanR
* Recipient (R) : TOP10 (with a TEM1 gene in chromosome)
* Conjugation time : 3 hours 30 min
* D.O. (600 nm) = about 1 (for recipient strain), about 0,5 (for donor/control strain)

![image](https://static.igem.org/mediawiki/2018/0/02/T--UCLouvain--project-result-16.png)

![image](https://static.igem.org/mediawiki/2018/b/b7/T--UCLouvain--project-result-7.jpeg)

Conjugation in plate test 1: On left : Donor and recipient strain after conjugation. On right : control and recipient strain after conjugation.

* First result seems to show that conjugation append (at least in control strain) and that donor strain (if have conjugated) seems to kill the AmpR gene as expected. This first test is non quantitative, so we don’t know exactly how
  much bacteria we have, and more test are needed.

#### Efficiency of Crispr/Cas9 system by conjugation.

To verify the effectiveness of our system using Cas9 carried by a mobilizable plasmid, we contacted the number of events or a recipient cell (TOP10 PyrF::TEM-1) lost its red phenotype when losing its plasmid pSB4K5\_bbaJ04450 after
conjugation with a donor cell (S17-1) containing the mobilizable plasmid containing Cas9 and an anti-RFP RNA guide. A negative control was performed using a donor strain that did not include guide RNA. Donor cells were counted after
dilution on LB kanamycin and recipients on LB ampicillin. Colony counts are carried out three times for each dilution. **The effectiveness of our system using Cas9 carried by a mobilizable plasmid give 27% for a D:R ratio 1:169.**

![image](https://static.igem.org/mediawiki/2018/b/b1/T--UCLouvain--project-result-12.png)

Table 3: Averages of the counts before and after conjugation and transfer of the mobilizable plasmid pK18mob\_CAS9.
Averages achieved over three spreads per sample. The interval is given by twice the standard deviation.

#### Conjugation in fluorescence microscopy

To visualize at the scale of a cell, the effect of the transfer of a mobilizable plasmid, containing Cas9 and its RNA anti RFP guide, by conjugation: we performed a 60-minute time lapse on a mixture containing the donor strain (S17-1 with
pK18mob\_CAS9\_sgRNA anti RFP and pSB4C5\_R0010\_E0840 (GFP)) and the recipient strain (DH5a with pSB4A5\_J04450 (RFP)).