Part:BBa_K195613

pCI with GFP generator

The composite part is made of standard lambda cI regulated promoter, RBS sequence, GFP gene and two terminators. This operon generates GFP and is sensitive to its repressor – cI.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 719
  

Usage and Biology

The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor BBa_C0051.

Repression analysis

cI binding results in repression of transcription. This feature was used to determine if this device is repressible by cI in vivo. To achieve this bacteria containing another device - a cI generator (BBa_K077039) were transformed with this device. After induction of cI expression with IPTG we measured decline of relative fluorescence produced by this device (full protocol: http://2015.igem.org/Team:Vilnius-Lithuania/Labjournal ).

Figure 1. GFP inhibition experiment. Relative fluorescence data normalized to sample with highest fluorescence (0.5 mM IPTG). (-) – Negative control (only BBa_K077039); 0.1 IPTG – 10 mM IPTG – samples with different IPTG concentrations. Error bars represent standard deviations of two biological replicates for each type of IPTG concentration.

We determined that this device is sensitive to cI (Figure 1). It can be seen that upon induction of cI expression there is a significant drop in relative fluorescence, the general tendency is that d fluorescence signal depletes with increasing concentrations of IPTG, however we see great variation in our data. Similar data was obtained during replications of this experiment. We suggest that BBa_K195613 is indeed sensitive to cI and the effects of cI on transcription levels from this promoter are somewhat titratable, however we claim that this system (BBa_K077039 + BBa_K195613) is highly unpredictable. We suggest that this unpredictability might be due to the low translation levels of cI protein (weak RBS) combined with its instability (LVA tail) and its nature of a dominant repressor. These effects combined might lead to an effect in which changes in a small population of repressor might have great effects on the whole system.