Part:BBa_K733018

xylR+PxylA+RBS+GFP+Double Terminator

This construct is built to measure the relative efficiency of xylose inducible promoter in E. coli. We intend to use GFP as an indicator to characterize this xylose inducible promoter derived from Bacillus megaterium (BBa_K733002). In our characterization, we will investigate the influence of concentration of xylose on xylose inducible promoter and use GFP intensity changes over certain period of time to represent the regulatory effect of this promoter in different xylose inducing conditions.

Characterization

Background Information [http://2012.igem.org/Team:HKUST-Hong_Kong/Module/Regulation_and_control (link to our Regulation and Control Module)]

The reason for using the xylose inducible promoter is to enable control on the expression of toxin and BMP2. Xylose is not toxic and normally not present in the human colon. This provides us an easy way to induce BMP2 expression without disrupting normal human body function.

Objective On characterization, we want to test whether the promoter works in E. coli DH10B strain and if it works, what is the absolute promoter activity under varied experimental condition (i.e. xylose concentration).

Intended Result

1. Xylose inducible promoter is functional in E.coli.

2. After the inducer concentration has reached certain level, a relatively stationary GFP expression level (expression upper-limit should be observed.

Method

The absolute promoter activity was measured with respect to xylose concentration.

The same reporter gene (BBa_E0240) was used to indicate promoter activity. E. coli carrying the right construct was cultured to log phase. Following the addition of xylose at various predetermined concentrations, at a time point around the mid-log phase, the GFP intensity and OD595 were measured for every 30 mins (up to 120 mins). Independent curves indicating the GFP intensity units (of various xylose concentrations) with respect to time were then plotted, following which the respective absolute promoter activities were calculated.

Characterization Procedure

1. Constructing xylR-PxylA-BBa_E0240-pSB1A2;

2. Preparing supplemented M9 medium (see below);

3. Culturing E. coli carrying xylR-PxylA-BBa_E0240-pSB1A2 and E. coli without constructs in supplemented M9 medium and measuring the growth curve respectively;

4. Culturing the above mentioned bacteria in supplemented M9 medium to log phase;

5. Adding xylose at different concentrations to different sets of bacterial culture;

6. Measuring the GFP intensity and OD595 values across time for every set of bacterial culture containing different xylose concentrations;

7. Plotting independent curves showing the GFP intensity units of various xylose concentrations with respect to time;

8. Plotting a graph to demonstrate the absolute promoter activity under different inducer concentrations;

9. Compiling the results.

Data Processing

1. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120 mins);

2. For GFP intensity, curve reflecting GFP expression change was plotted; for OD595, average value was taken;

3. GFP synthesis rate was then obtained by calculating the slope of linear regression line of the above mentioned curve;

4. Absolute promoter activity for the promoter under different inducer concentrations were calculated by dividing the corresponding GFP synthesis rate over the average OD595 value;

5. Averaged absolute promoter activity was then obtained by averaging the respective sets of absolute promoter activity values.

Result

1. Shown in the figure below, with the addition of xylose, GFP expression increased. This tells us that the xylose inducible promoter is functional in E. coli DH10B strain.

2. When no xylose was added, a limited amount of GFP was expressed. This suggests that the xylose inducible promoter is to some extent leaky.

3. A relatively stationary GFP expression level was observed at xylose concentrations of 1% to 5%. Despite some other variables (see discussion for more details), the data suggests that the minimum inducer concentration for triggering a full induction should lie somewhere between 0% and 1%.

4. For 10% inducer concentration, the GFP expression was relatively lower. There could be several reasons for this occurence, such as suboptimal growth conditions due to high osmotic pressure. (See discussion for more details.)

Discussion

1. It is quite obvious that addition of xylose induces GFP expression in this construct. However, a slight issue remains: even when no xylose was added, a minute but detectable amount of GFP was still expressed. This shows that the xylose inducible promoter is leaky. It should be noteworthy that this version of the xylose inducible promoter has undergone mutagenesis on 3 different sites on the repressive gene for standardization purposes. Even so, the mutagenesis is done while preserving an identical codon translation. As such, similar to the Ptms promoter, activity of the xylR-PxylA promoter might be different in E. coli from that of B. subtilis due to expression in a heterologous system. Further characterization on this promoter in B. subtilis would be our future aim as time was limited.

2. Another interesting fact to note is that the E. coli strain used is one in which it's xylose metabolic operon remains active. As such, one might assume that the observed GFP expression upper-limit ("stationary expression level"),particularly at 1% or 2% is not the "true" upper-limit, since there should be underlying metabolism of xylose. In order to eliminate this possible error, higher concentrations of xylose was used and the promoter activity does not appear to vary greatly, suggesting that the stationary expression level reflects the maximum promoter activity. While this problem is solved, due to the relatively large concentration difference of inducer, it appears that the maximum promoter activity was achieved at 1% xylose, implying that we are unable to determine the exact minimum inducer concentration requirement for maximum activity. Based on the current result, it is safe for us to make a conjecture that the minimum concentration required lies between 0% and 1% xylose.

3. For the decreased GFP expression at 10% xylose concentration, one possible reason is that the high osmotic pressure caused by the xylose in the medium may inhibit the growth and metabolism of bacteria, thus reducing the bacterial population and/or its GFP expression. Another possible, but unlikely reason could be that the over-expression of induced GFP expression may disturb the normal bacterial function, leading to a low overall GFP expression.

Supplemented M9 Medium Composition

1. 5X M9 Salt Composition (1L)

(1) 64g Na₂HPO₄﹒

(2) 15g KH₂PO₄

(3) 2.5g NaCl

(4) 5.0g NH₄Cl

2. Minimal 1X M9 medium (1L)

(1) 200ml of 5X M9 Salt

(2) 2ml of 1M MgSO₄

(3) 100μl of 1M CaCl₂

(4) 5ml of 40% glycerol

3. Supplement (for the final medium)

(1) 1mM thiamine hydrochloride

(2) 0.2% casamino acids

Improvement of Characterization by DTU-Denmark 2015

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 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

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 847
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2058

Improvement of Characterization by Aix-Marseille University 2024

Objective

The objective of our work was to measure the effect of a catabolic competitor more specifically glucose on the activity of the Pxyl promoter.

Method

The induction of the Pxyl promoter was investigated using the W3110 strain containing the plasmid pOK12 with the pXyla-GFP. Cultures were prepared from two 2YT growth media, with or without the addition of ampicillin at 100 µg/mL. The optical densities (OD) measured at 595 nm for the initial cultures were 7.6 for W3110 and 7.4 for the XXb6 strain.

Cultures were diluted tenfold to inoculate 2 mL of media, adding 27 µL of the cell dilution (ODi = 0.01). The incubation was carried out at 37°C with shaking at 200 rpm. Measurements of OD at 595 nm and fluorescence were taken in the stationary phase to assess the impact of different xylose concentrations on the expression of the GFP reporter gene.

Induction conditions varied with different xylose percentages (0%, 0.015%, 0.05%, 0.1%, 0.15%, 0.5%, and 1%), combined with the presence or absence of glucose. Volumes of xylose at 10%, 20%, and 50% were adjusted accordingly for each condition. For instance, for 0.5% xylose, 15 µL of 20% xylose solution was added, while for 1%, 40 µL of 50% xylose was used.

Characterization Procedure

Fluorescence measurements were performed using a Tecan reader at a single time point in the stationary phase. Each data point represents the average of two biological replicates, as the third replicate was not comparable.

Results

Analysis of Pxyl promoter induction by xylose revealed significant differences between conditions with and without glucose. In the absence of glucose, increasing xylose concentrations resulted in elevated fluorescence levels. The estimated KM was 33.2 ± 18.5 mM, indicating a low affinity of the promoter for xylose.

In contrast, in the presence of glucose, fluorescence was significantly reduced at xylose concentrations below 40 mM, demonstrating an inhibitory effect of glucose on promoter induction.

This phenomenon aligns with the principle of catabolite repression, where glucose, as the preferred carbon source, suppresses the utilization of alternative sugars such as xylose. At higher xylose concentrations, the inhibitory effect of glucose appeared to diminish, allowing fluorescence to approach levels observed without glucose.

Discussion

These findings highlight the importance of glucose concentration in regulating xylose-induced expression. Further investigations may be needed to explore the complex interactions between glucose and xylose (or other monosaccharide) in promoting the Pxyl activity, as well as to determine the implications of the estimated KM for optimizing induction strategies in future applications.