Part:BBa_K4159011:Design

pBAD and reversed P2 promoters

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 239
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 179
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

For ordering the part, some changes might be needed in the beginning of the sequence as the beginning is very TA heavy. A few GC nucleotides will help the production to succeed when you order the part as a whole.

Description:This part consists of two promoters pBAD, which is a continuous promoter, and of P2, which is an inducible promoter. We have placed the two promoters in two different direction, P2 being reversed and pBAD in the left to right direction. This way we try to avoid false positive results with the downstream genes, incase either promoter is a little "leaky". Between the promoter we have a spacer sequence and after both promoters we have placed the B0034 RBS sequence.

Characterization:

Figure 1. Amplification of part 1

The amplification of the BBa_K4159011 part went well after a few trials of PCR where we tested different annealing temperatures. Two bands were observed on the gel with a stronger band at 63 °C than 62 °C (Figure 1).

The amplified band had a concentration of 24.3 ng/µL.

The digest was observed as a strong band on the agarose gel (Figure 2)

Figure 2. Gel picture of digested part1

However, for some reason, the transformation of the fragment did not work. We tried different cells for the transformation, both alpha5 and top10 E.coli cells for the transformation, but with zero success in getting colonies. We also tried to longer the ligation time in case there that was the issue, and different ratios (3:1 and 5:1) of DNA to vector ratio. We also went back to the first step of PCR, and tried different annealing temperatures to optimize it to have an as high a concentration of the fragment for digestion and ligation as possible. For our final design (the submitted BBa_K4159011 part) we removed the 6bp spacer from the second draft and replaced it with a random sequence of 50bp. The random sequence was generated from the University of California’s Random DNA generator (http://www.faculty.ucr.edu/~mmaduro/random.htm). The GC content for the randomization was put as 0.5.

Figure 3. Transformed part1

After the changes, we succeeded transforming the part1 plasmid into the cells (fig.3). As this part was transformed into the same plasmid which BBa_K4159009 was already inserted in we wanted to verify that both parts were inserted correctly. We purified the plasmid from the cells and used both parts reverse and forward primer and ran a PCR.

Figure 4. Part 1 and part2 after amplification from the purified plasmid.

Figure 4. shows great results of both parts being inserted. As both parts were inserted with specific sticky end restriction sites, we believe that both parts are also inserted the correct way.

As the plasmid was purified from the cells, the cells also shifted in slight shade of green, which tells that the promoter part is inserted correctly and that the P2 promoter might be a little leaky even if it is not induced yet. To view the measuring results of the bioreporter construct visit our results page on wiki: https://2022.igem.wiki/aalto-helsinki/results

References: composite part, The part is taken partly from the iGEM part registry and can be found through the link: BBa_I746104 [taken 15.6.2022]. https://parts.igem.org/Part:BBa_K206000, pBAD strong promoter, iGEM parts registry [taken 15.6.2022]

Source

The part is taken partly from the iGEM part registry and can be found through the link: BBa_I746104 [taken 15.6.2022]. https://parts.igem.org/Part:BBa_K206000, pBAD strong promoter, iGEM parts registry [taken 15.6.2022]