iGEM 2020 — Team UGent2_Belgium

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# Team:UGent2 Belgium/Notebook

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# The Notebook

**Summary of important primers,****vectors****and their characteristics.**

At the MEMO group we already have plasmids available containing the naringenin production pathway. However, the pathway genes are expressed using the *E. coli*specific P22 promoter and are on the *E. coli*specific vector pSC101. Therefore, we need to test the functionality of the P22 promoter in *C. necator*to check if the genes will be expressed and transfer the pathway genes from pSC101 to the broad-host-range vector pBBR1-MCS2.

|  |  |  |  |
| --- | --- | --- | --- |
| **Strain** | **Temperature** | **Antibiotic** | **Info** |
| s5424 | 30 °C | Amp | Strain containing the P22 promoter |
| s5429 | 30 °C | Kan | Strain containing the Naringenin pathway |
| s5246 | 37 °C | Chl | Strain containing mKate2 |
| s6142 | 37 °C | Kan | pBBR1-MCS2 acceptor vector |

**Start by the end of****A****ugust**

**25/8**

Collecting templates:

Grow all strains with required plasmids overnight in 10 mL LB + 10 µL of the antibiotic necessary (1000x stock solution) at 30°C or 37°C, depending on the plasmid used, and 200rpm (LS-X AppliTek, Nazareth, Belgium) for plasmid purification.

|  |  |  |  |
| --- | --- | --- | --- |
| **Strain** | **Temperature** | **Antibiotic** | **Info** |
| s5424 | 30 °C | Amp | Strain containing the P22 promoter |
| s5429 | 30 °C | Kan | Strain containing the Naringenin pathway |
| s5246 | 37 °C | Chl | Strain containing mKate2 |
| s6142 ipv s6118 | 37 °C | Kan | pBBR1-MCS2 acceptor vector |

**26/8**

Collecting templates:

Plasmids were purified from grown cultures. The concentration of purified plasmids is given below:

|  |  |  |
| --- | --- | --- |
| Template | Concentration   (ng/µL) | Quality? |
| S5246 | 132.2 | OK |
| S5424 | 23.2 | OK |
| S5429 | 30.6 | OK |
| S6142 | 44.3 | OK |

Construction pBBR1MSC2\_P22\_RBS\_mKate2:

1. *Amplification of the**fragments**P22**\_RBS and mKate2**from the purified templates*
   Dilution template (1000x) and primers (100x), this setting is for all following PCR procedures unless stated otherwise.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Primers** | **Template (1000x)** | **Length (bp)** | **Polymerase** | **Name** |
| 9469 + 9470 | sMEMO5424 | 285 | PrimeSTAR HS | P22-RBS-lin |
| 9471 + 9472 | sMEMO5246 | 1015 | PrimeSTAR HS | mKate2-linNAR |

2. *Gel**electrophoresis**to evaluate**the PCR**.*

![](https://static.igem.org/mediawiki/2020/5/59/T--UGENT2_Belgium--a1.png)

Lane 1: P22-RBS-lin  => correct
Lane 2: mKate 2- lin (1) => Upper band between 1.0 and 1.2 kb. This is correct. However, the concentration is very low.

Lane 3: 2LOG NEB marker

Another PCR is performed on the product of mKate2-lin in order to obtain higher amounts of the fragment.

**27/8**

Construction pBBR1MSC2\_P22\_RBS\_mKate2:

1. *PS-PCR of mKate2-lin**(1)**product to achieve**a**higher amount.*

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Primers** | **Template** | **Fragment length****(bp)** | **Polymerase** | **Product** |
| **9471 + 9472** | Previous mKate2-lin (1) | 1015 | PrimeSTAR HS | mKate2-lin (2) |

2. *Gelelectrophoresis**of**PCR fra**gment**mKate**2**–**lin**:*

![](https://static.igem.org/mediawiki/2020/5/5f/T--UGENT2_Belgium--a2.png)

Lane 1: mKate 2 (2) - Lin => correct
Lane 2: 2LOG NEB marker

The gelelectrophoresis was done in a thick gel, and the staining with EtBr was not long enough.

**September**

**1/9**

Construction pBBR1MSC2\_P22\_RBS\_mKate2:

*1.**PCR Purification of the linear fragments and**DNA**concentration**determination*

|  |  |  |  |
| --- | --- | --- | --- |
| **Fragment** | **Conc****entrat****ion**   **(ng/µL)** | **Quality** |  |
| **P22-RBS-lin** | 87.2 | OK |  |
| **mKate****2 (1)-NAR** | 11.8 | NOK | Discard |
| **mKate****2 (2) - NAR** | 153.6 | OK |  |

2. *Run an overnight GGA assembly of the**fragments**P22**\_RBS**, mKate2**and its destination vector pBBR1MCS2.*

Pagina-einde

Construction pBBR1MSC2\_P22\_NarProduction pathway:

1. *GXL-PCR of the**naringenin production pathway genes*

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Primers** | **Template** | **Fragment length****(bp)** | **Polymerase** | **Product** |
| **9704 + 9705** | sMEMO5429 | 7017 | GXL | NarProduction pathway-lin |

**2/9**
Construction pBBR1MSC2\_P22\_RBS\_mKate2:

Electroporate 1 µL of the GGA mix in E. coli Top 10 cells, plate on LB + Kan and incubate overnight at 37 °C

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/6/6a/T--UGENT2_Belgium--a3.png)1. *Gelelectrophoresis**of G**X**L-PCR 1/9*

Lane 1: naringenin production pathway-lin
Lane 9: 2LOG NEB ladder

Aspecific interactions occurred during the GXL-PXR of naringenin production pathway; these interactions resulted in multiple linear fragments. However, the band of interest, at 7017 kb is the most visible.

2. *PCR purification**&**DNA concentration**determination:*

|  |  |  |
| --- | --- | --- |
| **Name** | **Concentration** | **Quality?** |
| **Nar production pathway** | 185.8 | OK |

3. *Overnight golden gate assembly**of the naringenin production pathway fragment with the destination vector pBBR1MCS2*

**3/9**

Construction pBBR1MSC2\_P22\_RBS\_mKate2:

Cheking transformants by cPCR and subsequent gelelectrophorese.

Purify pBBR1MSC2\_P22\_RBS\_mKate2 from positive clone and subsequent sequencing.

![](https://static.igem.org/mediawiki/2020/2/26/T--UGENT2_Belgium--a4.png)

Lane 1: pBBR1MCS2\_P22RBS\_mKate 2 (1) Lane 2: " " (2)
Lane 3: " " (3)

Lane 4: " " (4)

Lane 5: " " (5)

Lane 6: " " (6)

Lane 7: " " (7)

Lane 8: " " (8)

Lane 9: Log2 NEB marker

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Electroporate GGA assembly of pBBR1MCS2\_naringenin production pathway in *E. coli*TOP10 electrocompetent cells

Regenerate for 1 hour at 37°C

Plate on LB agar + Kan and incubate overnight at 30°C

**4/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Pick good colonies for colony PCR from the plates grown overnight. Two colonies were present on the plate and picked for colony PCR.

First attempt to perform a colony PCR and the primers used were 8529 and 5677. This should create a product of 656 bp.

**8/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/4/4e/T--UGENT2_Belgium--a5.png)1. *Gelelectrophoresis**of colony PCR*

Lane 1: 2logNEB
Lane 2: Naringenin pathway iGEM

Unfortunately, no product was obtained from the colony PCR.

**9/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

The retrial of picking up the linear fragments for the naringenin production pathway. Perhaps something went wrong during the GGA, and that might be the reason why the plasmid is not present in the colonies.

1. *Q5-PCR of naringenin pathway**-**lin*

Naringenin pathway

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Primers** | **Template (1000x)** | **Length (bp)** | **Polymerase** | **Name** |
| **9704 + 9705** | sMEMO5429 | 7017 | Q5 (60°C) | NarProduction pathway |

2. *Gele**ctroph**o**rese**of PCR fragment:*

![](https://static.igem.org/mediawiki/2020/f/f6/T--UGENT2_Belgium--a6.png)

Lane 1: Naringenin production pathway
Lane 2: Log2 NEB

Unfortunately, this Q5-PCR with annealing at 60°C did not give a linear fragment.

3. *3**.**Overnight**PrimeStart**GXL*

Another attempt to pick up the linear fragment containing the genes for the naringenin pathway.

Naringenin pathway

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Primers** | **Template (1000x)** | **Length (bp)** | **Polymerase** | **Name** |
| **9704 + 9705** | sMEMO5429 | 7017 | GXL | NarProduction pathway |

**10/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/e/e6/T--UGENT2_Belgium--a7.png)

*Ge**lelectrophoresis**of the overnight GXL*

Lane 9: naringenin production pathway
Lane 10: 2Log NEG ladder

Unfortunately, there is again no linear fragment present on the gel.

**15/9**Construction pBBR1MSC2\_P22\_RBS\_mKate2:

Sequencing of pBBR1MCS2\_P22\_RBS\_mKate2

Construction pBBR1MSC2\_P22\_NarProduction pathway:

1. *Theoretical**analysis of Naringenin pathway*

An in-silico testing of all the components involved in the naringenin pathway to see if something can be improved about the cloning.

Analysis Mix wizard, clone manager:

At 55°C Annealing -> 1 product formation. 7017 bp.

At 60°C Annealing -> 1 Product formation,7017 bp

At 65°C, no binding of 9705.

Highest annealing temperature that gives a linear fragment equals 62 °C.

3' end primer dimers present.

Primer analysis:

All good.

9704 => All good and Tm 78

9705 => All good and Tm 76

2. *Q5-P**CR (60°**C**annealing) Naringenin pathway*

**=> Add GC enhancer** because the Annealing temp calculated by clone manager is 12°c lower compared to the one suggested in NEB. And the text below said to reduce aspecific interactions to add GC enhancer.

Pagina-einde

3. *Gelelectroph**o**resis**of**naringenin production pathway fragment*

![](https://static.igem.org/mediawiki/2020/6/6e/T--UGENT2_Belgium--a8.png)

Lane 1: naringenin production pathway

Lane 2: Log2 NEB marker

The PCR result continued to improve, but a new problem arises. There are two bands present instead of the one band at 7017 bp desired. Purification of the band from the gels could be a solution. However, staining of the gel is standard with ethidium Bromide in the lab. Purifying the gel and taking it out of the contained environment poses significant risks and demands for multiple safety measures. So, it was decided to try to adapt the PCR reactions further.

**16/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Another attempt for trying to obtain a pure amplification of the naringenin pathway from the template: GXL 54 °C overnight PCR.

**17/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

*Gelelectrophoresis**of the overnight GXL**:*

![](https://static.igem.org/mediawiki/2020/e/e8/T--UGENT2_Belgium--a10.png)

Lane 1: 2log NEB marker
Lane 2: naringenin pathway

Again, there are multiple bands visible since nearly all the different possibilities have been tried, a new PCR based approach is necessary.

**22/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

The new approach consists of splitting up the naringenin production pathway. Splitting up might reduce the formation of two separate bands.

PCR of naringenin pathway in two parts => New approach.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Primers** | **Template (1000x)** | **Length (bp)** | **Polymerase** | **Name** |
| **9704 +****9739** | sMEMO5429 | 3783 | PS (55°C) | NarProduction pathway 1 - lin |
| **970****5****+****9738** | sMEMO5429 | 3260 | PS (55°C) | NarProduction pathway 2 – lin |

**23/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/6/63/T--UGENT2_Belgium--a11.png)1. *Gele**lectroph**o**resis**of**PCR fragments Nar 1 and Nar 2*

Lane 1: LOG2 NEB marker
Lane 2: Nar 1 (3783 bp) => correct
Lane 3: Nar 2 (3260 bp) => correct

2. *CPEC of**Naringenin**fragments formed by splitting up the naringenin pathway*

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Mix** |  | **Concentration (ng/µL)** | **Length (bp)** | **V (µL)** |
| **Q5 buffer** |  |  |  | 5 |
| **dNTPs** |  |  |  | 5 |
| **DMSO** |  |  |  | 1 |
| **Q5 polymerase** |  |  |  | 0.5 |
| **Nar pathway 1** |  | 72.7 | 3783 | 1.3755158184 |
| **Nar pathway 2** |  | 62.0 | 3260 | 1.3899192482 |
| **MQ** |  |  |  | 10.7345649333 |

3. *Overnight a GG**A assembly is performed**with the CPEC products of the naringenin fragments and the receiver vector pBBR1MCS2.*

**24/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Electroporation of GGA pBBR1MCS2\_naringenin production pathway in *E. coli*TOP10
Regeneration for 1 hour.
Growth on LB + Kan plates overnight

**29/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Colony PCR of pBBR1MCS2\_naringenin production pathway in *E. coli*TOP10. Primers chosen so amplified production contains all 4 genes.

|  |  |  |
| --- | --- | --- |
| **Mix** | **V 1 colony (µL)** | **V 8 colonies (µL)** |
| **mQ** | 11.325 | 101.925 |
| **Taq buffer** | 1.5 | 13.5 |
| **dNTPs** | 1.5 | 13.5 |
| **Taq polymerase** | 0.075 | 0.675 |
| **FW: 9172** | 0.3 | 2.7 |
| **RV: 7358** | 0.3 | 2.7 |

**30/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/8/80/T--UGENT2_Belgium--a12.png)1. *Gelelectrophoresis**of**pBBR1MCS2\_naringenin production pathway**colony PCR*

Lane 1-8: Colony PCR of pBBR1MCS2\_naringenin\_production pathway (4 genes, 7358 bp)

Lane 9: 2log NEB ladder

Perhaps the fragment was too long, and that leads to the failure of the colony PCR. The GGA should have worked out because of the excellent visibility of the bands present in the gel of the linear fragments.

2. *Second**attempt for a**colony PCR**of pBBR1MCS2\_naringenin production pathway*

Performed with another primer pair (overnight)

|  |  |  |
| --- | --- | --- |
| **Mix** | **V 1 colony (µL)** | **V 8 colonies (µL)** |
| **mQ** | 11.325 | 101.925 |
| **Taq buffer** | 1.5 | 13.5 |
| **dNTPs** | 1.5 | 13.5 |
| **Taq polymerase** | 0.075 | 0.675 |
| **FW: 5164** | 0.3 | 2.7 |
| **RV: 9541** | 0.3 | 2.7 |

4570 bp to be expected

**October**

**1/10**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/9/9c/T--UGENT2_Belgium--a13.png)1. *Gelelectrophoresis**of Col PCR pBBR1MCS2\_naringenin production pathway**:*

Upper gel: Lane 29-30: naringenin colony PCR
Lowest gel: Lane 1-6: naringenin colony PCR
Lane 7: 2log NEB

There is a smear present around the site where the fragment is expected, but it is not good enough to be sure.

2. *T**he**CPEC that was done before the GGA pBBR1MCS2\_naringenin production pathway was not necessary. It might be that this caused considerable burden on the GGA. So**,**the GGA pBBR1MCS2\_naringenin production pathway is redone starting from the linear fragments.*

**2/10**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Electroporation of the GGA assembly of pBBR1MCS2\_naringenin production pathway in *E. coli*TOP10
Regeneration for 1 hour.
Growth on LB + Kan plates overnight

**6/10**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Lien redid the GGA pBBR1MCS2\_naringenin production pathway with using a newly grown receiver plasmid pBBR1MCS2
Just to be sure that there is nothing wrong with the receiver template.

**7/10**

Evaluation of pBBR1MCS2\_P22\_RBS\_mKate2 functionality in *C. necator*, via growth trial:

1. *I**solate**cultures*

|  |  |
| --- | --- |
| **pBBR1MCS2\_P22****\_RBS\_****mKate2** | P22 vector |
| **pBBR1MCS2\_Aar 1** | Aar1 template |

Isolate culture on LB plate + kan + gent is grown overnight.

2. *C**ontrol of colonies via**cPCR**and**s**etting up the preculture**s**i**n**an MTP plate*

Stock solution of LB + Kan + Gent (10 mL LB + 10 µL Kan + 10µL Gentamicin ) in 15 mL falcon.

In the middle row of the MTP => Inoculation of the strains grown on a plate in a growth plate (transparent 96-well plate) using LB + antibiotics (150 µL in total). Pick colonies, for colony PCR and put it in the MTP. MTP plate is the backup plate of the colony PCR.

Keep 3 Blancs!

Overnight colony PCR of *C. necator* samples taken for growth trial, and GGA naringenin (2/10)

Fourth attempt for a colony PCR of pBBR1MCS2\_naringenin\_production\_pathway

|  |  |
| --- | --- |
| **Plasm****i****d** | **Primers** |
| **pBBR1MCS2\_naringenin\_production\_pathway** | 8432-8952 |
| **Template****naringenin\_production\_pathway** | 8432-8952 |
| **pBBR1MCS2\_P22\_RBS\_mKate2** | 9469-4206 |
| **pBBR1MCS2\_Aar 1** | 9451-9647 |

For pBBR1MCS2\_P22\_RBS\_mKate2 and pBBR1MCS2\_Aar 1 the backup plate is the MTP preculture plate. For each plasmid, 12 colonies were picked and inoculated.

For pBBR1MCS2\_naringenin\_production\_pathway and template naringenin\_production\_pathway, eight colonies were picked and incubated on a backup plate.

**8/10**

Construction pBBR1MSC2\_P22\_RBS\_mKate2:

![](https://static.igem.org/mediawiki/2020/1/13/T--UGENT2_Belgium--a14.png)1. *Gelelectrophoresis**of colony PCR*

Lane 1- 8: Naringenin template
Lane 9: 2log NEB ladder
Lane 10-17: Naringenin pathway (attempt Lien)
Lane 18: 2Log NEB ladder
Lane 19-28: pBBR1MCS2\_P22RBS from *C. necator*
Lowest gel
Lane 1-12: pBBR1MCS2\_Aar1 from *C. necator*.
Lane 13: 2Log NEB ladder

* Growth trial was cancelled due absence of the plasmid in the host (C. necator).

2. *Electroporation of P22 in C. necator*

electroporation in *C. necator*

2 hours regeneration

plate the C. necator cells on LB + Kan + Gent plates.

Construction pBBR1MSC2\_P22\_NarProduction pathway:

1. *Gelelectrophoresis**of colony PCR**(see figure above)*

2. *Electroporation of**pBBR1MCS2\_naringenin\_production\_pathway**(6/10) in E. coli TOP10*

electroporation in *E.coli*TOP10

1 hour regeneration

plate the TOP10 cells on LB + Kan plates.

**9/10**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

![](https://static.igem.org/mediawiki/2020/7/75/T--UGENT2_Belgium--a15.png)1. *C**olony**PCR**with**the**s**ame primers as in the fourth trial w**as**used.*
2. *Gelelectrophor**esis**of**pBBR1MCS2\_naringenin\_production\_pathway.*

Lane 1-4: Naringenin pathway
Lane 5: 2Log NEB
Lane 6-9: Naringenin pathway

**13/9**

Evaluation of P22 functionality in *C. necator*:

1. *Isolate**cultures*Isolateculture of pBBR1MCS\_P22\_RBS\_mKate2 in *C. necator*
   Overnight growth on LB + Kan

Construction pBBR1MSC2\_P22\_NarProduction pathway:

GGA of pBBR1MCS2\_naringenin\_production\_pathway

**14/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

Electroporation in *E. coli* TOP10

electroporation in *E.**coli TOP10*

1-hour regeneration

plate the TOP10 cells on LB + Kan plates.

**15/9**

Construction pBBR1MSC2\_P22\_NarProduction pathway:

1. colony PCR trial for pBBR1MCS2\_naringenin\_production\_pathway.

Again new primers are used for the colony PCR of pBBR1MCS2\_naringenin\_production\_pathway.

|  |  |
| --- | --- |
| **Mix** | **V 8 colonies (µL)** |
| **mQ** | 101.925 |
| **Taq buffer** | 13.5 |
| **dNTPs** | 13.5 |
| **Taq polymerase** | 0.675 |
| **FW: 9469** | 2.7 |
| **RV: 4206** | 2.7 |

2. Gelelectrophoresisof colony PCR

![](https://static.igem.org/mediawiki/2020/6/64/T--UGENT2_Belgium--a16.png)

Upper gel
Lane 1-8: pBBR1MCS2\_naringenin pathway
Lane 9: 2Log NEB marker
Lane 10-15: pBBR1MCS2\_P22\_RBS\_mKate2 in *C. necator* col pcr
Lower gel
Lane 1-2: pBBR1MCS2\_P22\_RBS\_mKate2 in *C. necator* col pcr
Lane 3: 2LOG NEB marker

Evaluation of P22 functionality in *C. necator*:

1. 1*.**Col**ony**PCR to check pBBR1MCS\_P22\_RBS\_mKate2 in C. necator**.*

|  |  |
| --- | --- |
| **Mix** | V 8 colonies (µL) |
| **mQ** | 101.925 |
| **Taq buffer** | 13.5 |
| **dNTPs** | 13.5 |
| **Taq polymerase** | 0.675 |
| **FW: 8432** | 2.7 |
| **RV: 8952** | 2.7 |

2. *Gelelectrophoresis**of colony PCR (see picture above)*

**20/10**

Evaluation of P22 functionality in *C. necator*:

1. *Isolate**cultures*

Preculture pBBR1MCS\_P22\_RBS\_mKate2 in *C. necator* and *E. coli*TOP10. . necator with pBBR1MCS2\_AaRI is used as negative control. Isolate colonies taken from an existing plate.

2. *Control of colonies via**cPCR**and**s**etting up the preculture**s**i**n an MTP plate*
   Loading order of MTP plate:
   3x pBBR1MCS\_P22\_RBS\_mKate2 *C.**necator* (col 1)*,*3x pBBR1MCS\_P22\_RBS\_mKate2*E.coli*(col 1), 3x AaR I *C. necator*(all biological replicates)*,*3x blank

3x pBBR1MCS\_P22\_RBS\_mKate2 *C.**necator* (col 2)*,*3x pBBR1MCS\_P22\_RBS\_mKate2 **E.coli** (col 2), 3x AaR I *C. necator*(all biological replicates)*,*3x blank

Colonies were checked before.

**21/10**

Evaluation of P22 functionality in *C. necator*:

1. Start growth trialpBBR1MCS\_P22\_RBS\_mKate2 in  *E.* coli  TOP10, empty template vector Aar1 and blank.

The cells did not grow :(

2. *Isolate cultures*

Isolate colonies picked from isolate culture plate of pBBR1MCS\_P22\_RBS\_mKate2 in *E. coli*K12 MG1655, pBBR1MCS2\_AarI in K12 MG1655. All are taken as biological triplicates.

3. *S**etting up the preculture**s**i**n an MTP plate*

Preculture start of pBBR1MCS\_P22\_RBS\_mKate2 in *E. coli*K12 MG1655.

pBBR1MCS2\_AarI in K12 MG1655

Blanco.

All in triplicates

**22/10**

Evaluation of P22 functionality in *C. necator*:
*1. Setting up the final MTP plate*

Take 20µL from the precultures and add it to 140 µL defined medium in an MTP plate. This plate is called the dilution plate.

Take 10µL from the diluted preculture and add it to140 µL defined medium in the final plate

Loading order:

pBBR1MCS2\_P22mKate2 in K12 MG1655, three biological triplicates

pBBR1MCS2\_AarI in K12 MG1655 (= s6127), three biological triplicates.

Blanco, three triplicates.

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# Team:UGent2 Belgium/Engineering

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* [Home](https://2020.igem.org/Team%3AUGent2_Belgium)
* Team
  + [Members](https://2020.igem.org/Team%3AUGent2_Belgium/About_Us)
    [Experts](https://2020.igem.org/Team%3AUGent2_Belgium/Experts)
    [Attributions](https://2020.igem.org/Team%3AUGent2_Belgium/Attributions)
    [Collaborations](https://2020.igem.org/Team%3AUGent2_Belgium/Collaborations)
    [Contribution](https://2020.igem.org/Team%3AUGent2_Belgium/Contribution)
* Project
  + [Description](https://2020.igem.org/Team%3AUGent2_Belgium/Description)
    [Model](https://2020.igem.org/Team%3AUGent2_Belgium/Model)
    [Implementation](https://2020.igem.org/Team%3AUGent2_Belgium/Implementation)
    [Proof Of Concept](https://2020.igem.org/Team%3AUGent2_Belgium/Proof_Of_Concept)
    [Sponsors](https://2020.igem.org/Team%3AUGent2_Belgium/Sponsors)
* Lab
  + [Notebook](https://2020.igem.org/Team%3AUGent2_Belgium/Notebook)
    Engineering
    [Protocols](https://2020.igem.org/Team%3AUGent2_Belgium/Protocols)
    [Primers](https://2020.igem.org/Team%3AUGent2_Belgium/Primers)
    [Safety](https://2020.igem.org/Team%3AUGent2_Belgium/Safety)
* [Human Practices](https://2020.igem.org/Team%3AUGent2_Belgium/Human_Practices)
  + [Human Practices](https://2020.igem.org/Team%3AUGent2_Belgium/Human_Practices)
    [Education](https://2020.igem.org/Team%3AUGent2_Belgium/Education)
* [Medals](https://2020.igem.org/Team%3AUGent2_Belgium/Medals)

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  ### Hi! I'm Bubbly, can I help you?

  [click here](#top)  to go to the top

  [click here](#prod)  to go to production of naringenin

  [click here](#edible)  to go to the edible pearl

  [click here](#bottom)  to go to the bottom

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# Team:UGent2 Belgium/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UGent2 Belgium/Results

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)