Part:BBa_J100534:Design

Constitutive RFP Promoter in E. coli

Assembly Compatibility:

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

  Illegal NheI site found at 35
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

We decided not to order the AHL chemical because there is no lux cassette in the E. coli cultures that we will use (not possible for the lux gene to be expressed). This lux R gene is typically expressed in other bioluminescent bacteria, including Vibrio fischeri. Therefore, we are allowing it to function as a constitutive promoter. We have one experimental group that will be compared to a negative control, which contains water instead of the diluted 50 nM oligo solution, and a positive control, which contains neither water nor the oligo solution but DNA that produced RFP. GGA was used to insert the promoters into the experimental groups (GGA master solution, ligase, oligo, and BSA I type II restriction enzyme were added and held at different temperatures). The BSA I restriction enzyme cut DNA at the restriction enzyme sites (located in the multiple cloning site of the plasmid) asymmetrically, creating "sticky ends" which allowed the promoter to be inserted. Ligase joined the promoter to the plasmid. We grew colonies from each of the three groups, and then we extracted three red cells from the experimental (X) groups to grow overnight colonies X1, X2, and X3. Because we could work with the AHL/lux repressor system, we decided to alter temperature as a variable. We hypothesized that allowing the bacteria to incubate for one hour at a temperature of 42 degrees Celsius will act as a repressor for the specific promoter and RFP production. We chose 42 degrees Celsius as the temperature so that we did not denature or effect the growth rate of our E. coli colonies. We incubated 200 micrometers of 40nM X group samples. We used 200 micrometers of the same 40nM X samples that did not receive heat treatment as the control. We then ran them in the spectrophotometer to compare the amount of fluorescence produced with the optic density of both the incubated and non-incubated groups. We created a ratio of red to optical density for the purpose of measuring the amount of red (RFP produced) in each cell. PCR was used to determine if the promoters were successfully cloned. PCR was done by adding PCR master mix (primer, DNA polymerase, dNTPs, and a buffer) to N and X1, X2, X3 groups. The bacteria provided the template for PCR. PCR results were run through gel electrophoresis to compare sizes. The added primer will be shorter (no longer than 60 nucleotides plus sticky ends) than the original primer in the N group that produced GFP. In moving forwards, we are currently looking into whether or not there is a gene that is similar to the lux R gene that is naturally expressed in E. coli that our promoter might be able to affect/function with. The promoter is also repressed by the quorum sensing autoinducer AHL. To see if E. coli altered with this promoter can divide in the same way, we will take a dense cell colony, dilute it, and centrifuge it down. We will take an aliquot from the top of the centrifuged sample, which should theoretically contain some chemical that induces quorum sensing division. We will then spread that on another cell colony of the same dilution and see if it alters RFP production.

Source

This promoter sequence came from an article published on NCBI: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444344/