Difference between revisions of "Team:Bielefeld-CeBiTec/Results/unnatural base pair/preservation system"

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The <i>E. coli</i> strain BL21(DE3) was transformed with the plasmid pSB3C5. LB<sub>Cm25</sub> plates were incubated overnight at 37 °C.  
 
The <i>E. coli</i> strain BL21(DE3) was transformed with the plasmid pSB3C5. LB<sub>Cm25</sub> plates were incubated overnight at 37 °C.  
 
Three colonies were picked and incubated in 150 mL LB media supplemented with LBCm25 overnight at 37 °C. Well-plates with 12, 24 and 48 wells were inoculated to an OD<sub>600</sub> of 0.1 with a total of three biological replicates. Three different cultivation volumes were used to observe the cell growth in the different well plates with the different volumina.  
 
Three colonies were picked and incubated in 150 mL LB media supplemented with LBCm25 overnight at 37 °C. Well-plates with 12, 24 and 48 wells were inoculated to an OD<sub>600</sub> of 0.1 with a total of three biological replicates. Three different cultivation volumes were used to observe the cell growth in the different well plates with the different volumina.  
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Revision as of 19:44, 29 October 2017

Retention System

Pretests

Due to the high costs of our UBPs we had to carefully plan the experiments involv-ing these bases. Therefore, we carried out cultivation experiments of Escherichia coli BL21(DE3) in micro well plates, because performing experi-ments in a microscale would contribute significantly to a lower cost of the experi-ments. To cultivate at the ideal growth conditions, we performed pretests. In our ex-periments, we tested for the ideal culture volume, surface size and shaking frequency. The E. coli strain BL21(DE3) was transformed with the plasmid pSB3C5. LBCm25 plates were incubated overnight at 37 °C. Three colonies were picked and incubated in 150 mL LB media supplemented with LBCm25 overnight at 37 °C. Well-plates with 12, 24 and 48 wells were inoculated to an OD600 of 0.1 with a total of three biological replicates. Three different cultivation volumes were used to observe the cell growth in the different well plates with the different volumina.


Table 1: Used well plates and the different cultivation volumes in the particular plate. Three replicates per plate and volume were recorded at an rpm of 350.
12 Well Eppendorf 0030721012 24 Well Eppendorf 0030722019 48 Well Eppendorf 0030723015
1 mL 1 mL 0.5 mL
2 mL 2 mL 0.75 mL
3 mL 3 mL 1 mL
The cultivation was carried out in the VWR – Incubation Microplate Shaker and the OD600 measured via NanoDrop ND-1000 Spectrophotometer. Three technical replicates were measured for each sample.

Figure 1:
A: Average OD600 of the three biological replicates for each cultiva-tion volume in the 12 well plate over the cultivation period. B: Average OD600 of the three biological replicates of each volume in the 24 well plate over the cultivation period. C: Average OD600 of the three bio-logical replicates of each cultivation volume in the 48 well plate over the cultivation period.

Figures 1 shows that cultivation is possible in all well plates. The lowest OD600 is consistently achieved by the highest volume with values at 2.271, 2.027 and 1.286 using the 12 well plate, 24 well plate and 48 well plate, respectively. The highest OD600 is associated with the lowest volume with values of 2.629, 2.416 and 1.889 using the 12 well plate, 24 well plate and 48 well plate, respectively. Irrespective of volume, the highest OD600 values were reached using the 12 well plate. Specifically, the OD600 value of 2.271 using 3 mL in the 12 well plate is still higher than the OD600 value of 2.027 using 1 mL in the 24 well plate. After determining the best plate and volume to perform cultivations with, we investigated the influence of the rpm by cultivating three biological replicates at 500, 600, and 700 rpm in the 12 well plate in 1 mL.

Figure 2:
Growth curves of the three replicates of E.coli BL21 (DE3) in LBCM25 at 350, 500, 600, and 700 rpm, respectively. The DNA concentration was measured under the optimal condition of 600 rpm and 1 mL volume.

Cultivation at 600 rpm yields the highest OD600 with a value of about 4.961 after 6.5 h. If the rpm is further increased, the cells grow badly due to an out of phase movement of the media. We finally concluded that the perfect cultivation conditions are reached in a 12 well plate with a total volume of 1 mL and shaking with 600 rpm. For analysis of the retention and preservation of the unnatural base pair, the isolation of a sufficient amount of plasmid DNA from the culture is important. In order to evaluate the plasmid concentration during the cultivation, we cultivated at 600 rpm on a 12 well plate in 1 mL. We inoculated the wells with a starting OD600 of 0.1 and conducted a plasmid isolation using 1 mL for each measurement. The highest DNA concentration was reached in the exponential phase with about 85 ng/µl in 30 µl.