Difference between revisions of "Team:NTNU Trondheim/Results"

Biofilm Assay Results

• Isolation of pdCas9 and pgRNA from Top10 cells

  The plasmids were ordered from Addgene and delivered in E. coli Top10 cells. We started by plating the cells on agar plates containing the selective antibiotics for the specific plasmids – Chloramphenicol for selection of pdCas9 and ampicillin for pgRNA. The plates were incubated overnight, and single colonies were picked and inoculated in LB medium with the respective antibiotics. We isolated the plasmids by MiniPrep and checked the purity of the isolates with NanoDrop. The results from the NanoDrop are given in Table 1. Since the 260/230-ratio is less 2.00 (eller 1.80 😊), it may indicate the presence of contaminants which absorbs strongly at 230 nm. Nevertheless, both the concentrations and the 260/280-ratios indicate that there was enough DNA to continue the process for both replicates of pdCas9 and pgRNA.

  Table 1: ...

  Sample (replicate)	Concentration [ng/µL]	260/280-ratio	260/230-ratio
  pgRNA (1)	62.3	1.86	1.41
  pgRNA (2)	68.1	1.84	1.26
  pgCas9 (1)	20.1	1.96	1.41
  pgCas9 (2)	52.1	1.79	1.05

  The isolated plasmids were digested with the restriction enzyme BspHI and the plasmid fragments were run on an agarose gel alongside GeneRuler 1 kbp ladder. A digestion tool in the software Benchling was used to predict the fragment lengths and gel patterns for each of the plasmids after cutting with BspHI. The pdCas9 was expected to be cut into two fragments with lengths of 2741 and 3964 bp, and the pgRNA was expected to be cut into three fragments with lengths of 105, 1008 and 1451 bp. As seen in Figure 1, both replicates for pgRNA seem to have bands with the expected lengths of approximately 100, 1000 and 1500 bp. The band at 100 bp seemed to be only vaguely visible, possibly due to the fragment’s short length, which renders it to only bind a low amount of dye. For pdCas9 only replicate 2 had the correct bands at approximately 2700 and 4000 bp.
  

  

  
• Plasmid transformation into competent DH5α cells

  The isolated pdCas9 and pgRNA from the E. coli Top10 cells were separately transformed into competent E. coli DH5α cells. The transformed cells were streak plated, and single colonies were picked and inoculated the next day. The OD₆₀₀ values of the overnight cultures were approximately 0.1 (pdCas9: 1.23; pgRNA: 0.88). The cell cultures were miniprepped and nanodropped. The NanoDrop results, given in Table 2, implied that both pgRNA replicates and pdCas9 replicate 2 might have been RNA contaminated.

  Table 2: ...

  Sample (replicate)	Concentration [ng/µL]	260/280-ratio	260/230-ratio
  pgRNA (1)	60.2	2.09	2.26
  pgRNA (2)	44.2	2.18	2.84
  pgCas9 (1)	194.1	1.56	0.73
  pgCas9 (2)	53.4	2.25	3.53

  Test digest was performed on the isolated plasmids using the restriction enzyme BspHI. The gel imaging is shown in Figure 3. Both plasmids gave the expected number of bands and fragment lengths (pgRNA: 100, 1000 and 1500 bp; pdCas9: 2700 and 4000 bp). This observation verified that the transformation had been a success. Interestingly, replicate 1 of dCas9 seemed to give the right restriction pattern in this time (for comparison see Figure 1). Nevertheless, we still decided to only continue using replicate 2 of this plasmid since the reason for the dissimilarities were uncertain.

  

• Overhang Extension PCR

  

  Overhang Extension PCR was performed to insert the 20 bp anti-luxS tracker sequence into the pgRNA. This sequence is complementary to a specific location near the TBS in the luxS gene, which is a gene involved in the production of the quorum sensing molecule Autoinducer-2. Primers with the anti-luxS overhang were designed using the software Benchling (see Design for more details about Overhang Extension PCR). The PCR products were run on an agarose gel to separate the linearized plasmid with the inserted of anti-luxS from the original circular pgRNA without anti-luxS. Figure 4 shows the gel separation of the PCR products. The strong band at 2500 bp corresponds to the linearized plasmid. The circular configuration of the original pgRNA lacking the anti-luxS, makes it coil up and migrate further in the gel than the linearized plasmid due to less resistance. The original pgRNA can be seen at approximately 1500 bp.

  
  

  The linearized plasmid at 2500 bp was excised from the gel, purified and nanodropped. The concentration was 52.8 ng/µL and the 260/280- and the 260/230-ratio was 1.90 and 0.27, respectively. All though the DNA concentration and 260/280nm-ratio were acceptable, the low 260/230-ratio indicated that the recovered plasmids probably were contaminated. The contamination might be remnants of substances in the PCR-mix, agarose gel and/or chemicals used during the purification step.

  
  

  The linearized pgRNA with the inserted anti-luxS sequence was transformed into

  Table 3: ... Sample (replicate) OD₆₀₀ Concentration [ng/µL] 260/280-ratio 260/230-ratio pR2 (1) 0.95 37.2 2.06 4.29 pR2 (2) 1.00 123.2 1.77 1.01
  

  The transformation was verified by test digest with PstI. PstI was chosen because it has a restriction cut site in the anti-luxS sequence. The band pattern obtained after gel electrophoresis corresponded with the expected lengths of the plasmid fragments of 490 bp and 2094 bp (see Figure 5). The transformation with pgRNA and insertion of the anti-luxS sequence was considered successful.

  
  

  

  

• Double transformation of

  E. coli

  DH5α and TG1

  The pdCas9 and the pgRNA with anti-luxS were double transformed into competent E. coli DH5α and TG1 cells and plated on agar plates with both chloramphenicol and ampicillin. These cell strains were selected due to their different capacities to produce biofilm – DH5α being a poor biofilm producer, whereas TG1 is known to produce an abundancy of biofilm components if the conditions allow. Cultures of each of the transformed strains were miniprepped and the isolated plasmids were nanodropped and digested with PstI and BamHI-HF.

  
  

  The NanoDrop results for the isolated plasmids from DH5α and TG1 are given in Table 4. For DH5α the 260/280-ratio was higher than 2.00 for both replicates, which may indicate that our isolates might have contained RNA impurities. The 260/230-ratio for the first replicate seemed acceptable, but the ratio for replicate 2 seemed oddly high. This gives reasons to believe that this sample was contaminated or was highly diluted. In addition, bobbles were detected when measuring this replicate. This might have interfered with the result giving a higher ratio than the actual value. The plasmids isolated from TG1 were all deemed good for further use, based on their near optimal ratios.

  
  

  Table 4: ...

  Sample (replicate)	Concentration [ng/µL]	260/280-ratio	260/230-ratio
  DH5α, pR2L + pC2 (1)	219.9	2.01	1.82
  DH5α, pR2L + pC2 (2)	35.3	2.03	3.86
  TG1, pR2L + pC2 (1)	24.7	1.88	2.34
  TG1, pR2L + pC2 (2)	23.0	1.87	2.18
  TG1, pR2L + pC2 (3)	28.3	1.86	2.34
  TG1, pR2L + pC2 (4)	29.4	1.76	1.34
  TG1, pR2L + pC2 (5)	26.9	1.74	2.19