Part:BBa_K4737007

pLtetO

pLtetO promoter which can be repressed by tetR protein well, regulates the expression of LacI repressor.

Usage and Biology

In the safety module we have designed “logical suicide circuit” to maintain two plasmids in the project and to induce engineered bacteria to commit suicide through intake of doxycycline after the end of the mission (more details see in design). The “logical suicide circuit” involve two strong and tightly repressible promoters pLacO and pLtetO. To validate the “logical suicide circuit”, we first tested the functionality of the pLacO and pLtetO promoters.

Figure 1. Schematic representation of the "logical suicide circuit"

1. Validation of pLacO and pLtetO
Aiming to test the functionality of the pLacO and pLtetO promoters, we constructed the pET-GFP-LacI and pJKR-L-tetR plasmids. The pET-GFP-LacI plasmid utilized the constitutive promoter prpsM to express the LacI protein, which effectively represses the pLacO promoter. Meanwhile, pLacO regulates the expression of GFP. Similarly, the pJKR-L-tetR plasmid contains a TetR repressor protein that efficiently represses the pLtetO promoter, with pLtetO governing the expression of GFP. We separately transformed the pET-GFP-LacI and pJKR-L-tetR plasmids into BL21(DE3) cells to validate a portion of our logical circuit. (called BL21-GFP-lacI and BL21-GFP-tetR).

Figure 2. Construction of plasmid pET-GFP-LacI and pJKR-L-tetR plasmids to test pLacO and pLtetO

The engineered bacteria BL21-GFP-lacI was cultured at 37°C, 220 rpm until OD₆₀₀=0.5, and original BL21 as a blank control operate accordingly. Subsequently, 0.1 mM IPTG was added to the solution of BL21-GFP-lacI bacteria, followed by culturing for 6 h at 37℃, 220 rpm. A negative control consisting of BL21-GFP-lacI (IPTG-free) was also cultured under the same condition. Then we measured the GFP fluorescence intensity of these cultures. The fluorescence intensity of BL21-GFP-lacI+IPTG was found to be significantly higher than that of BL21-GFP-lacI and blank control, indicating that pLacO functions effectively(Figure 3). Similarly, BL21-GFP-tetR was cultured at 37°C, 220 rpm until OD₆₀₀=0.5, and original BL21 as a blank control operate accordingly. Subsequently, we add 0.01 μg/mL, 0.1 μg/mL, 0.2 μg/mL, 0.3 μg/mL doxycycline (Dox) into BL21-GFP-tetR separately and culture for 6 h at 37℃, 220 rpm. BL21-GFP-tetR (Dox-free) was cultured accordingly as a negative control. Measure the GFP fluorescence intensity of these cultures. The fluorescence intensity of BL21-GFP-tetR+Dox are significantly higher than BL21-GFP-tetR and blank control (especially BL21- GFP-tetR+0.1 μg/mL Dox) which means pLtetO functions effectively and the best concentration of Dox is about 0.1 μg/mL(Figure 3).

Figure 3. The fluorescence intensity of BL21-GFP-lacI with/without IPTG and BL21-GFP-tetR with/without Dox

2. Validation of Hok-Sok In order to further validate "logical suicide circuit" and test the function of Hok-Sok, we constructed two plasmids, pET-hok-sok-lacI and pGEN-prpsm::tetR. These two plasmids were then transformed into BL21(DE3) competent cells together. The pGEN-prpsm::tetR plasmid utilized the constitutive promoter prpsM to express the TetR protein. Within the pET-hok-sok-lacI plasmid, the pLtetO regulates the expression of LacI repressor, the promoter pLacO regulates the expression of antitoxin Sok and the trp promoter regulates the toxin Hok. However, it was later discovered that BL21(DE3) endogenously express LacI protein making it difficult to validate "logical suicide circuit" in this strain.

Figure 4. Construction of plasmid pET-hok-sok-lacI and pGEN-prpsm::tetR

Therefore, we tested pET-hok-sok-lacI plasmid alone. When pET-hok-sok-lacI works alone, toxin Hok and LacI repressor will be constitutively expressed. LacI will repress the expression of antitoxin Sok and leads to the death of engineered bacteria. The addition of IPTG, which can bind with LacI and inhibit its repression Sok production, preventing engineered bacteria from being killed by Hok.
The pET-T7-hok-sok-lacI plasmid was transformed into BL21(DE3) (BL21-Hok/Sok). BL21-Hok/Sok can grow in the LB-agar plates with IPTG as expected. We selected nine colonies and individually transferred them to 10 μL LB liquid medium. Subsequently, the bacteria solution was transferred to the LB-agar plates without IPTG. They were cultured at 37℃ for 36 h. It was found that all of the colonies can’t grow up which means pET-T7-hok-sok-lacI can work well as we designed. However, it is a pity that we don’t have enough time to validate complete "logical suicide circuit" in our chassis strain.

Figure 5. Bacterial BL21-Hok/sok can grow in the LB-agar plates with IPTG(left), but cannot live in the LB-agar plates without IPTG(right).

3. Plasmid Stability Test
To assess the stability of plasmids without selection pressure, we constructed the pET-RFP-lacI-Kan plasmid. Afterwards, the pET-RFP-lacI-Kan plasmid was transformed into BL21(DE3) along with the pGEN-Amp plasmid, which is the skeleton of functional plasmid pGEN-prpsm::tetR. The pET-RFP-lacI-Kan is the skeleton of functional plasmid pET-hok-sok-lacI with a reporter gene RFP.

Figure 6. Plasmid construction of pET-RFP-lacI-Kan and pGEN-Amp

The engineered bacteria (BL21-Kan/Amp) were cultured in the LB liquid medium without antibiotics for 48 h and 72 h. And then we found that 5.1% of BL21-Kan/Amp lost pET-RFP-lacI-Kan while none of BL21-Kan/Amp lost pGEN-Amp in 48h. And 15.4% of the BL21-Kan/Amp lost pET-RFP-lacI-Kan while 1.07% of BL21-Kan/Amp lost pGEN-Amp in 72 h. These data have been provided to model.

Figure 7. a, BL21-Kan/Amp colonies of 48 h and 72 h in LB-agar plates without antibiotic. Red colonies are engineered bacteria containing pET-RFP-lacI-Kan while white lost pET-RFP-lacI-Kan. b, A certain number of single colonies were randomly picked from non-antibiotic LB-agar plates (a) to LB liquid medium containing ampicillin and grown in a 37℃ incubator. H10, H11 and H12 are LB liquid medium without colonies as blank control. Measure the OD₆₀₀ of these culture and compare to blank control to find out the lost rates of pGEN-Amp.

To calculate the total number of generations of engineered bacteria by culturing time, we measured the specific growth rates of original BL21(DE3) and BL21-Kan/Amp. It revealed that engineered bacteria grow slower than original bacteria due to the burden of the exogenous plasmids. The maximum specific growth rates of engineered bacteria decrease 15.9% to original bacteria. These data have been provided to model.

Figure 8. The specific growth rates of original BL21(DE3) (left) and BL21-Kan/Amp (right). We measured the specific growth rates by measuring the OD600 of the samples at 15 minutes intervals. The maximum specific growth rates were measured in the exponential growth time.

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]