Team:UGent2 Belgium/Notebook

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 August 

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 thefragments 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 

 

  1. Gelelectrophoresisto evaluate the PCR. 

 

 

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) 

 

 

 

 

 

 

 

 

  1. Gelelectrophoresisof PCR fragment mKate 2 – lin: 

  

 

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 

Concentration 

(ng/µL) 

Quality 

 

P22-RBS-lin 

87.2 

OK 

 

mKate 2 (1)-NAR 

11.8 

NOK 

Discard 

mKate 2 (2) - NAR 

153.6 

OK 

 

 

  1. Run an overnight GGA assembly of thefragmentsP22_RBS, mKate2 and its destination vector pBBR1MCS2. 

 

Pagina-einde 

Construction pBBR1MSC2_P22_NarProduction pathway:  

 

  1. GXL-PCR of thenaringenin 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:  

  1. Gelelectrophoresisof GXL-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. 

  1. PCR purification& DNA concentration determination: 

Name 

Concentration 

Quality? 

Nar production pathway 

185.8 

OK 

 

  1. Overnight golden gate assemblyof 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. 

 

 

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:  

  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 

 

  1. Gelectrophoreseof PCR fragment: 

 

Lane 1: Naringenin production pathway 
Lane 2: Log2 NEB 

 

 

 

 

 

 

 

 

 

 

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

  1. 3.OvernightPrimeStart 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:  

Gelelectrophoresis 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. 

 

 

15/9Construction pBBR1MSC2_P22_RBS_mKate2:  

Sequencing of pBBR1MCS2_P22_RBS_mKate2 

 

Construction pBBR1MSC2_P22_NarProduction pathway:  

  1. Theoreticalanalysis 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 

 

  1. Q5-PCR (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 

  1. Gelelectrophoresisof naringenin production pathway fragment 

 

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: 

 

 

 

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 

9705 + 9738 

sMEMO5429 

3260 

PS (55°C) 

NarProduction pathway 2 – lin 

 

23/9 

Construction pBBR1MSC2_P22_NarProduction pathway:  

  1. Gelelectrophoresisof 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 

 

 

 

 

 

  1. CPEC ofNaringenin 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 

 

  1. Overnight a GGA assembly is performedwith 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:  

  1. Gelelectrophoresisof 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.  

  1. Secondattempt for acolony 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:  

  1. Gelelectrophoresisof 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. 

  1. TheCPEC 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. Isolatecultures

 

pBBR1MCS2_P22_RBS_mKate2 

P22 vector 

pBBR1MCS2_Aar 1 

Aar1 template 

 

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

  1. Control of colonies viacPCR and setting up the precultures in 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 

Plasmid 

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:  

  1. Gelelectrophoresisof 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). 
  1. 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. Gelelectrophoresisof colony PCR (see figure above) 

 

  1. Electroporation ofpBBR1MCS2_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:  

  1. ColonyPCR with the same primers as in the fourth trial was used. 
  2. Gelelectrophoresisof 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. IsolateculturesIsolateculture 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 

 

  

  1. Gelelectrophoresisof colony PCR  

  

 

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.ColonyPCR 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 

 

  1. Gelelectrophoresisof colony PCR (see picture above) 

20/10 

Evaluation of P22 functionality in C. necator: 

  1. Isolatecultures

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. 

 

  1. Control of colonies viacPCR and setting up the precultures in 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 :( 

  1. 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.  

 

  1. Setting up the preculturesin 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.