Characterisation of xylR

Background
Experimental Design and Methods
Results
Discussion and Conclusion
References

Background
Experimental Design and Methods
Results
Discussion and Conclusion
References

Scroll down for more

Background

The xylose inducible promoter BBa_K733018 was of interest to our group as a means to control expression of a toxin that would function as a counter-selective marker for marker free integration of large pieces of DNA into Bacillus subtilis. Our idea was to use it to select for transfer of the 40.8kb tyrocidine operon from Brevibacillus parabrevis to Bacillus subtilis. The limitation of this repressor and promoter control system is that, as in E. coli, the repressor is leaky [1]. When used as a control for expression of toxins, it is necessary that the repressor is tight. Bhavsar et al. identified an alternative xylose inducible promoter from Bacillus subtilis W23 that showed tighter repression in Bacillus subtilis than the promoter from B. megaterium (BBa_K733018). Lin et al. used this improved repressor to control expression of the native E. coli toxin in Bacillus subtilis. Using this system, they were able to do marker-less gene deletions. We investigated the repressor activity in E. coli with the intention of using it for selecting for transfer of large gene clusters.

Experimental Design and Methods

We designed three different xylose inducible promoter constructs. The optimized B. subtilis repressor binding site in the promoter sequence was either replaced downstream of BBa_K733018 or the promoter sequence of BBa_K733018 was deleted. (Note: Two different size deletions were tested). Sequence for the optimized promoter was obtained from Lin et al. (part of sequence for mini-mazF cassette, ) and ordered as a gblock.

Generation of promoter constructs

The gblock and BBa_K733018 were amplified with primers containing 20-25 overhangs with plasmid or synthesized repressor, respectively. The two pieces of DNA were assembled by Gibson assembly according to the manufacture’s protocol. Transformants were selected and an overnight culture started. Plasmids were purified from overnight cultures and digested with restriction enzymes to confirm the correct insert.

Primers

	Primers for amplification of BBa_K733018	Primers for amplification of optimized repressor (gblock)	Restriction enzymes used to confirm insert
Construct 1 (entire promoter sequence replaced)	5’- AGGATCTGGTtagtttattggataaacaaactaactcaattaagat-3’ 5’- AGACTTGATATGggttattattcaaattgcagatcaagct-3’	5’- tgaataataaccCATATCAAGTCTTTCATGAAAAACTAAAAAAAATATTGAAA-3’ 5’- ccaataaactaACCAGATCCTCCTTTAGATGCA-3’	NsiI + PstI
Construct 2 (part of promoter replaced)	5’- AGGATCTGGTatgcgtaaaggagaagaactt-3’ 5’- AGACTTGATATGggttattattcaaattgcagatcaag-3’	5’- tgaataataaccCATATCAAGTCTTTCATGAAAAACTAAAAAAAA-3’ 5’- ctttacgcatACCAGATCCTCCTTTAGATGC-3’	NsiI + PstI
Construct 3 *new promoter right upstream of translation start)	5’-GGAGGATCTGGTatgcgtaaaggagaagaacttttcactg-3’ 5’-AGACTTGATATGtgtgactctagtaccctttgatttaagtgaac-3’	5’- ctagagtcacaCATATCAAGTCTTTCATGAAAAACTAAAAAAAAT-3’ 5’- ctttacgcatACCAGATCCTCCTTTAGATGCAT-3’	NsiI + PstI

Biolector M2P-Labs

Of each of the three constructs #1 was tested once, #2 was tested 3 times, and #3 was tested 5 times for GFP expression on the Biolector. The Biolector is a plate based reader that can simulate optimal growth conditions with temperature, shaking, and humidity control while measuring absorbance and fluorescence for measureable GFP and Biomass readings. E. coli transformants with BBa_733018 in the pSB1C3 plasmid were grown in 1.5 mL of liquid LB media with 6 ug/mL chloramphenicol (CAM) with or without 1% Xylose to release the XylR and induce GFP transcription and translation.

Protocol:

Measure the OD₆₀₀ of overnight cultures of transformants grown in LB media with 6 ug/mL chloramphenicol in order to prepare a dilution scheme so all samples have an initial OD₆₀₀ of 0.1
Prepare LB media with 7.5 ug/mL chloramphenicol. Take 100 mL of that sample and add 1.25 g of xylose to prepare a 1.25% xylose stock of LB media with 7.5 ug/mL of CAM.
Dilute overnight cultures in sterile deionized H₂O so that with a 1.25x dilution they will have an OD₆₀₀ of 0.1.
Transfer 1.2 mL of LB media with CAM +/- xylose to a 48 well Biolector flowerplate and add 300 uL of diluted overnight cultures. Assay was prepared in triplicate with a negative control
Cover Biolector plate with a gas permeable seal and place in Biolector chamber with an incubation temperature of 37°C, shaking at 1000 RPM, humidity at 95%, O₂ at 20.95%, Biomass absorbance at 620 nm, and GFP filter with fluorescence excitation at 488 nm and measurement at 520 nm. Measurements were taken every 10 minutes for up to 15 hours.

Results

All transformants were tested in triplicate and the standard deviation is included in the error bars on the graph. The absorbance readings were averaged and corrected for background by subtracting the values of the media only samples. It is interesting to note that all samples grown in 1% xylose had a higher amount of biomass and indicates that E. coli can use it as a nutrient source. This appears to verify the hypothesis of {link to previous group’s wiki} regarding utilization of xylose as a nutrient source. GFP expression appears to begin after 0.5 hours of growth and reaches a maximum rate of synthesis by 2.5-3 hours of growth.

Figure 1 Biomass Absorbance readings measured at 620 nm were unstable for the initial two hours of the experiment so the Biomass measurements start at 2.2 hours in this graph. All samples had an initial OD600 of 0.1 at the start of the experiment.

Figure 2 Average GFP measurements corrected for the background of the media at an excitation of 488 nm and absorbance of 520 nm

Discussion and Conclusion

The optimized promoter for expression control in Bacillus subtilis did not have the proposed function in E. coli. Whether this is the same in Bacillus subtilis was not tested in this experiment, but according to previous findings, the promoter is optimized for control in B. subtilis [?,?]. While we set out to improve a BioBrick function, we were only able to improve the description of the existing BioBrick by providing more detailed data for induction GFP synthesis from BBa_K7330018 in a 1% xylose solution.

References

Bhavsar, A, Zhao, X, Brown, E. Development and Characterization of a Xylose-Dependent System for Expression of Cloned Genes inBacillus subtilis: Conditional Complementation of a Teichoic Acid Mutant. Appl. Environ. Microbiol. January 2001 vol. 67 no. 1 403-410.

Team:DTU-Denmark/Description