Part:BBa_K625000

RBS-LacIM1

LacI_M1 (Bba_K625000) is a LacI variant with the same protein sequence as the wild-type LacI, but with a completely codon-modified DNA sequence. This enables to use both sequences on two different plasmids (with two different promoters) without recombination happening between them. This feature can be used to establish a bandpass filter.

Experimental setup

Figure 1: Design for the LacI and LacI_M1 characterization

To check the functionality of Bba_K625000, we engineered a test system for our BioBrick by adding a lac-dependent GFP reporter. A tet-promoter was put in front of lacI_M1 and TetR constitutively produced, thus preventing lacI_M1 from expression.

In the non-induced state of the system, lacI_M1 expression is repressed and the cells produce GFP. If anhydrotetracycline is added, TetR is released from P_tet and LacI is produced. LacI inhibits gfp transcription and the fluorescence decreases. Addition of IPTG although leads to the opposite case: LacI gets inhibited and the fluorescence increases.

The test system for Bba_K625000 was established as a single plasmid system. In the experiments we used the two different plasmids pSB1AK3 and pSB3C5 as backbones of the designed network. Experiments were performed in E.coli strain JM101, which was grown in M9 medium until exponential growth and then induced with increasing amounts of anhydrotetracyline (0 ng/mL, 1 ng/mL, 100 ng/mL) and IPTG (0 µM, 1 µM, 10 µM, 100 µM, 1000 µM). The cells were incubated in a 96-well plate at 37°C and every 15 minutes OD₆₀₀ and GFP fluorescence was measured.

Results

• pSB3C5
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Figure 2: time course of fluorescence depending on different expression levels of the transcriptional regulator LacI_M1. pSB3C5 was used as plasmid backbone for the test system.

• pSB1AK3
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Figure 3: time course of fluorescence depending on different expression levels of the transcriptional regulator LacI_M1. pSB1AK3 was used as plasmid backbone for the test system.

In general the test system for characterization of our BioBrick parts BBa_K625000 respectively BBa_K625001 worked as expected. In both system we can see that the fluorescence decreases with increasing amounts of anhydrotetracycline. This indicates that LacI_M1 is induced and thus represses the expression of gfp. By addition of IPTG LacI_M1 gets inhibited and therefore the GFP response increases. This tendency can be observed both in figure 1 and figure 2.

At a concentration of 100 ng/mL anhydrotetracycline, only a slight increase in fluorescence can be observed for increasing IPTG concentrations. We are supposing that the expression of lacI_M1 is fully induced at this level and thus the IPTG amount present in the system is not sufficient anymore to inhibit all of the LacI_M1.

In the case where no anhydrotetracycline is present in the medium, there can still be observed a change in fluorescence depending on the level of IPTG. The same pattern of induction can be observed here as in the weakly induced systems. The reason for this is likely due to the leaky expression of the lacI_M1 gene, resulting in a small amount of LacI_M1 always present in the cells and thus inhibiting the GFP production.

After about 300 min for pSB3C5 and about 200 min for pSB1AK3, the cells enter stationary phase and the fluorescence intensity and OD₆₀₀ do not increase anymore.

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
  COMPATIBLE WITH RFC[1000]