Team:BHSF ND/Project/Leakage
2019 IGEM BHSF_NDLeakage
Background
Gene expression depends largely on transcription and translation. Such processes display activated/non-activated states. For some situations, we would expect zero expression of GOI without the presence of inducer. However, leakage still exists due to the incomplete OFF stage of the transcriptional system. Even without proper inducers, GOI is still partly expressed in some cases. Such leakage is called the basal leakage. In some cases, as long as one molecule of toxin protein is translated, it can kill the entire cell. Thus, preventing leakage is vital in regulating gene expression. Current methods of solving leakage problems includes introducing strong inducible promoters to the system. However, there are substantially no regulating system or transcriptional factor that can function as a technically ON/OFF switch to prevent basal level leakage. This may be caused by weak RNAP-DNA interactions or different transcriptional capacities and induction rate of different promoters and inducers. 【1】
Design
We mainly focused on two types of inducible transcriptional promoter—XylS and pBAD. The XylS protein is the positive transcription regulator of the TOL plasmid meta-cleavage pathway operon Pm. XylS belongs to the AraC family of transcriptional regulators and exhibits an N-terminal domain involved in effector recognition, and a C-terminal domain. 【2】The activator XylS is usually activated by the benzoate and its derivatives (3Mbz in our design).
The pBAD promoter holds great potential to control the expression of genes that require tight regulation, as it is capable of both an induction and repression. The pBAD promoter exhibits an almost all-or-none behavior upon induction with arabinose when located on a high copy vector, but allows for gradual induction when cloned into a low copy vector.【3】 Based on these research findings, a low copy vector was used to investigate the ability to inhibit gene expression subsequent to induction of pBAD.
As the result shows, both system exhibit relative high level of fluorescence compared to our backbone design( BN006) which suggest the two inducible transcription system function properly.
Figure 1: XylS(BN007 contains BBa_K3202031) induction curve at different concentrations after 4 hours compared to backbone.(BN006 contains BBa_K3202029)
Figure 2: pBAD(BN009 contains BBa_K3202031) induction curve at different concentrations after 4 hours compared to backbone(BN006 contains BBa_K3202029)
Experiment & Results
Experiment & Results
In our experiment of leakage testing, we used low-copying plasmid backbone PSB4K5, with a PSC101 origin of replication.
Two inducers with their respective promoters (Arac & pBAD; Xyls & pm) are coupled with sfGFP to see if there is actually expression leakage when inducer is present quantitatively through flow cytometry. We tested fluorescence under different inducer concentration at the given time of 240 minutes and under different time stage at a given inducer concentration. Theoretically when inducers (Arac and 3MBz ) are present, promoters (pBAD and pm) are initiated therefore both sfGFP are expressed; while sfGFP shouldn’t be expressed if inducer is absent.
When measured at the end of 240 minutes without inducer,BN009( contains BBa_K3202031) has a basal leakage of 968 a.u. compared to BN006( contains BBa_K3202029). BN007(pBAD+sfGFP) also showed a leakage of 90 a.u. The result suggested that both system have noticeable leakage and XylS has much higher leakage.
When we gradually increased inducer concentration to 1mM, the fluorescence of BN006 stays at a constant level, whereas the fluorescence of both XylS and pBAD have increased exponentially. This is another sign that both system function properly.
Figure 3: Time induction curve for XylS(BN007 contains BBa_K3202030) at the concentration of 1mM compared to backbone (BN006 contains BBa_K3202029)
We found a intriguing result when measuring the fluorescence of the two system under a constant inducer concentration of 1mM. During the first 160 minutes, the fluorescence of BN007( contains BBa_K3202030)is 200 a.u. higher than BN006. However, at 240 minutes, the fluorescence value suddenly increased to 7641 a.u., which is 7590 a.u., higher than the backbone. Same thing happened to BN009. Therefore, in the subsequent experiments, systems are induced to up to 4 hours to ensure the accuracy of the experiment. Results shown above verified the presence of expression leakage of both systems when inducer is not expressed through the detectable fluorescence of reporter protein using flow cytometry.
Figure 4: Time induction curve for pBAD(BN009 contains BBa_K3202031) at the concentration of 1mM compared to backbone (BN006 contains BBa_K3202029)
Discussion
Through our design and experimental validation, we demonstrate the existence of leakages and the need to address leaksage. We tried to use a more efficient promoter, but it didn't solve the leakage. The leakage-related experiment drove us to design an efficient zero-leakage module to put toxin and ultimately form the design of the suicide system.
Reference
【1】Creecy & Conway, 2015
【2】https://2013.igem.org/Team:Peking/Project/BioSensors/XylS
【3】http://parts.igem.org/Part:BBa_I0500