Design
ALKR-PalkM system in the original organism
Figure 1. Mechanism of ALKR-PalkM system in Acinetobacter baylyi ADP1
Figure 2. Homologous modeling figure of protein ALKR
Protein ALKR and promoter alkM are originally found from Acinetobacter baylyi ADP1. The ALKR-PalkM gene circus is part of the regulation system of alkane metabolism.
Acinetobacter sp. are ubiquitous bacteria in natural aquatic and soil environment that are frequently found to be capable to degrade a broad range of carbon chain alkenes and alkanes[3]. And Acinetobacter baylyi ADP1 is able to use long-chain-length alkanes with at least 12 carbon atoms as the sole source of carbon and energy[4]. In Acinetobacter baylyi ADP1, P-alkM is the promoter of alkane hydroxylase genes which encodes alkane hydroxylase(alkM), a crucial enzyme in the degradation of alkanes. AlkM, together with rubredoxin (RubA) and rubredoxin reductase(RubB) forms a three-component alkane monooxygenase complex which oxidate inert alkane to the respective primary alcohol[4]. AlkR is an AraC/XylS-like transcriptional regulatory protein. It includes C-terminal DNA-binding domain for promoter binding and N-terminal domain for inducer recognition[2]. Its function is to recognize alkane molecule after its own dimerization and induce promoter alkM. The activity of alkane hydroxylase could impede ALKR’s function. When alkane molecules occur, they could be recognized by protein ALKR. A three component complex is formed. The inducer complex can bind with promoter alkM and activate the genes in the downstream of PalkM.
Our first trail
Our goal is to build a specific alkane sensing device in the cell. The device could sense alkanes and transfer the inconspicuous alkanes to some conspicuous signals, such as some chromophoric or florescent signals. We could place both alkane biosynthesis module and AlkSensor into E coli. The alkane molecules produced by alkane biosynthesis module can be recognised by AlkSensor. And AlkSensor generate a conspicuous signal which can be easily detected.
Figure 3. A rough idea of our design
Based on the ALKR-PalkM mechanism in Acinetobacter baylyi ADP1, we designed an alkane sensor in E coli. The sensor is composed of two parts: alkane sensing part and signal generation part. The core of alkane sensing part in Acinetobacter baylyi ADP1 is promoter alkM, so we took PalkM into our design. Besides, PalkM must co-function with transcription factor ALKR, so we constructed ALKR into the sensor. ALKR and PalkM are sufficient for sensing alkanes. The function of signal generation part is to output a conspicuous or easily detectable signal. In this case, we chose RFP as the reporter.
Our first design is shown in figure 3. We linked ALKR with a constitutive promoter,a strong promoter J23100, and linked RFP with P-alkM. AlkR is followed by a short terminator B1006.The two gene pieces are constructed into plasmid pSB1C3. The inputs of AlkSensor are alkanes and the outputs are RFPs.
Figure 4. The first version of AlkSensor
Optimization and our final construction
We performed a blank test on the first version of AlkSensor. Without any inducers, AlkSensor still shown significant leakage. We can see from the results that many of the bacterial colonies are red enough to be distinguished by naked eyes.
Figure 5. The leakage of the first version of AlkSensor is non-ignorable
Followings are three possible causes of the leakage:
a) Promoter alkM has some leakage, to be more specific, it binds to RNA polymerase without undergoing a conformation change and activates the transcription.
b)Protein ALKR or dimerized ALKR could induce promoter alkM without alkane molecules.
c)The terminator upstream of PalkM is BBa_B1006 which is relatively short (only 34 bp), and it has a weak terminating ability, while the constitutive promoter BBa_J23100 upstream of AlkR is strong, so expression of RFP might be influenced.
We Reconstruct AlkSensor:
a) Changed the promoter of ALKR to a weaker promoter.
b) Switch the position of gene pieces ALKR and PalkM-RFP.
c) Replace terminator B1006 with a stronger terminator, B0015.
Figure 6. AlkSensor after reconstruction
Through another blank test we found that the reconstruction could decrease AlkSensor’s leakage significantly. After optimization, the percent of bacterial colonies are red enough to be distinguished by naked eyes decrease from 95% to 15%.
Figure 7. Comparison of two versions of AlkSensor
We also used full dose octane to induce the two sensors. After a certain period of time of cultivation, the relative fluorescent intensity was measured. The results show that after reconstruction the leakage decreases by 50%, the dynamic range increases by 5 folds, which means that the optimized AlkSensor has stronger ability to distinguish different inputs.
Figure 8. Octane induce test of two versions of AlkSensor
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