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

Revision as of 19:58, 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. LB_Cm25 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 LB_CM25 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.

codA

Deletion of the codA gene could be useful for our project due to its ability to transform isoG and isoC into uracile. In this case the concentration of the UBPs decreases and our plasmid carrying the UBPs could not be replicated properly. We designed three compatible constructs for the potential deletion of codA following the protocol of Jiang et al.

At first we designed our construct carrying our synthesized single guide RNA (sgRNA), which is essential for the targeting specificity. Because of the presence of the sgRNA, Cas9 could carry out a sequence-specific, double-stranded break. In the case of a repair via homology directed repair with our designed PCR-construct, codA gets deleted. Just like Jian et al., we chose pTarget as our vector. Therefore, we annealed the sgRNA oligos, cloned them into the pTarget backbone via golden gate assembly and selected positive clones via blue-white screening.

The PCR-construct responsible for the homologous recombination, and therefore the codA deletion, was generated by two PCRs. In a first step, 700 bp upstream and downstream of codA were amplified. These two fragments were then inserted into the backbone using Gibson Assembly. The homologous recombination of the neighboring sequences of codA with the plasmid bound flanks leads to a deletion of this gene.

Figure 3:
Design of the flanking sequences for the deletion of codA.

As the Crispr-Cas9 system plasmid, we conceptualized pCas, also used by Jiang et al., containing the lambda-Red system for a higher recombineering efficiency. For the codA gene deletion you need to generate E. coli BL21 DE harboring pCas, and Arabinose (10 mM) has to be added for the generation of electocompetent E. coli BL21 DE for the lambda-Red induction. For the homology directed repair, one needs to transform pTarget and the PCR fragment into the pCas harboring E. coli. Then, one needs to verify positive transformants by colony PCR and DNA sequencing.

To cure pTarget, one can inoculate a positive colony at 30 °C for 8 to 16 hours in media containing kanamycin (50 mg L^-1) and IPTG (0.5 mM). For the curing of pCas, the cell can be grown overnight at 37 °C nonselectively. The challenging part of this is the biological meaning of codA because of its important role in the purine synthesis pathway. To counteract this, it is advised to supplement with uracile (Mahan et al., 2004).

@@ Line 119: / Line 119: @@
 <div class="contentbox">
      <div class="content">
-         <h3> Literature Workshop </h3>
+         <h3> <i>codA</i> </h3>
          <!-- Normaler Text -->
          <article>
-<i>codA</i>
 Deletion of the <i>codA</i> gene could be useful for our project due to its ability to transform isoG and isoC into uracile. In this case the concentration of the UBPs decreases and our plasmid carrying the UBPs could not be replicated properly.
 We designed three compatible constructs for the potential deletion of <i>codA</i> following the protocol of Jiang et al.
+<br></br>
 At first we designed  our construct carrying our synthesized single guide RNA (sgRNA), which is essential for the targeting specificity.  Because of the presence of the sgRNA, Cas9 could carry out a sequence-specific, double-stranded break. In the case of a repair via homology directed repair with our designed PCR-construct, <i>codA</i> gets deleted. Just like Jian et al., we chose pTarget as our vector. Therefore, we annealed the sgRNA oligos, cloned them into the pTarget backbone via golden gate assembly and selected positive clones via blue-white screening.
+<br></br>
 The PCR-construct responsible for the homologous recombination, and therefore the <i>codA</i> deletion, was generated by two PCRs. In a first step, 700 bp upstream and downstream of <i>codA</i> were amplified. These two fragments were then inserted into the backbone using Gibson Assembly. The homologous recombination of the neighboring sequences of <i>codA</i> with the plasmid bound flanks leads to a deletion of this gene.
@@ Line 140: / Line 145: @@
          <article>
            As the Crispr-Cas9 system plasmid, we conceptualized pCas, also used by Jiang et al., containing the lambda-Red system for a higher recombineering efficiency.
-For the <i>codA</i> gene deletion  you need to generate E. coli BL21 DE harboring pCas, and Arabinose (10 mM) has to be added for the generation of electocompetent <i>E. coli</i> BL21 DE for the lambda-Red induction. For the homology directed repair, one needs to transform pTarget and the PCR fragment into the pCas harboring E. coli. Then, one needs to verify positive transformants by colony PCR and DNA sequencing.
+For the <i>codA</i> gene deletion  you need to generate <i>E. coli</i> BL21 DE harboring pCas, and Arabinose (10 mM) has to be added for the generation of electocompetent <i>E. coli</i> BL21 DE for the lambda-Red induction. For the homology directed repair, one needs to transform pTarget and the PCR fragment into the pCas harboring <i>E. coli</i>. Then, one needs to verify positive transformants by colony PCR and DNA sequencing.
+<br></br>
 To cure pTarget, one can inoculate a positive colony at 30 °C for 8 to 16 hours in media containing kanamycin (50 mg L<sup>-1</sup>) and IPTG (0.5 mM). For the curing of pCas, the cell can be grown overnight at 37 °C nonselectively.
-The challenging part of this is the biological meaning of <i>codA</i> because of its important role in the purine synthesis pathway . To counteract this, it is advised to supplement with uracile (Mahan et al., 2004).
+The challenging part of this is the biological meaning of <i>codA</i> because of its important role in the purine synthesis pathway. To counteract this, it is advised to supplement with uracile (Mahan et al., 2004).
          </article>