Team:Mingdao/Improve
Improvements
IMPROVE EXISTING PARTS
Part Name [Number]:
- Pcar, synthetic promoter repressed by CRP [BBa_K861171]
- RBS + PhlF repressor + terminator [BBa_K1725041]
- PhlF repressible promoter + strong RBS + GFP [BBa_K1725001]
Triggering the suicide circuit
In our case of sugar hijacking, ideally, the tiny, living “agents” are supposed to kill themselves in the story to make a happy ending. Therefore, we introduced a glucose responsive elements and a repressor circuit to be connected to suicide genes (lysis and NucA).
Promoter Pcar [BBa_K861171] is a glucose responsive promoter created by WHU-China in 2012. Pcar promoter region was de novo designed with overlapping of CRP and RNA polymerase binding site. The initiation of transcription by RNA polymerase may be hindered by the binding of CRP, which occurs at the formation of cAMP-CRP complex in the low concentration of glucose. In other words, when the amount of glucose is high enough, Pcar would be turned on after the leaving of CPR due to the low concentration of cAMP, and vice versa.
PhlF repressor system contains the repressor PhlF [BBa_K1725041] and the PhlF repressible promoter [BBa_K1725001] created by Glasgow in 2015. PhlF could repress GFP fluorescence intensity by 83-fold according to the study of Glasgow’s work.
Lysis gene [BBa_K117000] created by NTU-Singapore in 2008 encodes Lysis protein which could not only lyse bacterial cell membrane but also activate the endonuclease of Colicin E7 (ColE7). The lysis-colicin is one class of bacteriocins which are produced to response to worsening environmental conditions and outcompete other bacteria.
NucA [BBa_K1159105] created by TU-Munich in 2013 from Staphylococcus aureus produces a thermostable exo- and endo-nuclease that is able to degrade genomic DNAs2. NucA also has a role in the cleavage of extracellular DNAs and preventing biofilm formation.
We’ve innovated this year a novel glucose responsive repressor system (Pcar-wRBS-PhlF-T-Pr-sRBS-GFP/pSB1C3 [BBa_K2230012]) by connecting these two system and extend the function of them.
To achieve our goal, we added the suicide genes of lysis and NucA in the back of GFP (Pcar-PhlF-T-Pr-GFP-Lysis-NucA/pSB1C3 [BBa_K2230017]) which are controlled under the same repressible promoter and hope to see the activity of suicide genes could respond against the presence of glucose.
Please go to DEMONSTRATION section below to check the function of this device in our experimental results.
DEMONSTRATION
To improve and extend the function of BioBrick devices of glucose responsive promoter and PhlF repressor system. We designed and created the glucose responsive repressor system and tested it in response to various concentration of glucose. GFP intensity was measured at Ex/Em = 488nm/518nm as reporter showing the response of repressible promoter in the presence of glucose.
Further, we also designed and created the glucose responsive suicide circuit and examined it in response to decreasing concentration of glucose. OD600 and cell numbers were calculated to understand the killing efficacy of suicide circuit in the loss of glucose.
The bacteria carrying the indicated vector were cultured in LB media supplemented with 34 μg/ml of chloramphenicol (Cm) at 37°C overnight. The next day, OD600 was measured and adjusted to 2.5 in M9 minimal media with various concentrations of glucose. The bacteria then were incubated for 4 hours at 37°C. GFP or OD600 were indicators of promoter activities as mentioned. For suicide assay, the culture media were further taken out and diluted 106 times following by spreading onto LB Cm agar plate at 37°C overnight. The third day, the numbers of colonies were counted and bacterial viability was calculated.
Positive (GFP expression control) : Pr-GFP/pSB1C3 [BBa_K1725001]
rGFP (Glucose repressive system): Pcar-PhlF-T-Pr-GFP/pSB1C3 [BBa_K2230012]
rSuicide (Glucose repressive system) : Pcar-PhlF-T-Pr-GFP-Lysis-NucA/pSB1C3 [BBa_K2230017]
Experiment & Result
Glucose responsive suppressor system
In the assay for repressor system, the data in Fig. 2 gave the similar results as team Glasgow did in 2015, in which the strong activity of the repressible promoter was significantly repressed in the presence of PhlF repressor.
Furthermore, we modified the expression of PhlF under the glucose responsive promoter (Pcar-PhlF-T-Pr-GFP/pSB1C3 [BBa_K2230012]). And the E. coli carrying this plasmid cultured in LB broth overnight was transferred to M9 minimal media with decreasing concentrations of glucose. The result in Fig 3 clearly indicated that the GFP activity driven by the repressible promoter was gradually increased in response to the loss of glucose to 1.88 folds compared to the initial GFP activity at the beginning culture in M9 media, suggesting that the level of expression of PhlF was positively corresponding to the concentration of glucose.
Glucose responsive suppressor suicide circuit
As you can see in Fig 2., the OD value in response to the decreasing concentration of glucose was gradually reduced to 1.89 much less than average 2.71 in control group without suicide gene expression, implying that the suicide proteins killed the cells in the loss of glucose in the environment. Moreover, when the bacteria were grown in M9 media with 0.5mM glucose for 4 hours, the survival rate was decreased to 34% compared to 56% of bacteria without suicide genes (Fig. 3). And the cell numbers were reduced to 671 compared to 1120 of bacteria without suicide genes. Both data confirmed that this suicide device works well and indicated that killing process began when glucose in the media was running out.
Discussion
The device of glucose responsive repressor system works in our study very well in response to increasing concentrations of glucose which controls the level of expression of PhlF repressors through glucose responsive promoter.
The suicide circuit connected the combination of repressor system and glucose responsive promoter were successfully demonstrated in response to the presence of glucose in the environment. We not only confirmed the data of iGEM previous work but also improve the existing parts by extending the function and application of biobricks.
© iGEM Mingdao 2017. Design: Kevin Li. All rights reserved.
