MOPS with glucose or glycerol?

After a deep analysis of CysE expression levels, toxicity, and production of cysteine we determined that the perfect medium to our purposes was MOPS supplemented with glucose. Below it is illustrated the road that brought us to this conclusion.

To better control protein expression, we used two different recipes of the MOPS minimal medium: one with glucose as the carbon source (MOPS B), the other with glycerol (MOPS A).

Glucose is an inhibitor of the araC-pBAD promoter, while glycerol does not have a comparable inhibitory mechanism. We have tested the effect of these two different MOPS medium on cells growth and cysteine production with the intention of determining the best growth protocol for our future applications on the statues.

To this purpose, we have first analyzed the amount of protein produced in the two media by looking at fluorescence intensity using our sfGFP tagged reporter of CysE (BBa_K731040).

Figure 3: Fluorescence intensity of CysE-sfGFP (BBa_K731040) after induction with arabinose (ara). MOPS supplemented with glycerol (A) is shown in blue, MOPS supplemented with glucose (B) is shown in yellow.

Protein levels obtained in the medium supplemented with glycerol were significantly higher than those obtained in MOPS supplemented with glucose because of the inhibitory mechanism of glucose.

In glycerol, thus, cells are producing a lot of CysE. To assess if and how CysE production is affecting cell growth, we perfomed serial dilutions at 8h after induction.

However, to our surprise cysteine production behaved differently. We tested cysteine production of cells samples uninduced and induced overnight in the two different media.

Figure 4: Ninhydrin assay on NEB10β expressing Part BBa_K731030. A representative replicate is shown. From left to right samples are: CysE in MOPS A before induction, CysE in MOPS A not induced grown overnight, CysE in MOPS A induced grown overnight, CysE in MOPS B before induction, CysE in MOPS B not induced grown overnight, CysE in MOPS B induced grown overnight. MOPS supplemented with glycerol (A), MOPS supplemented with glucose (B).

Surprisingly cysteine production was higher in the uninduced sample grown in glycerol. In glucose the situation was the opposite and we had high cysteine production in the induced sample. This effect was contrasting with protein expression levels that we observed. We then hypothesized that high expression of CysE could have a toxic effect on the cells, which could explain why in glycerol we observed a lower cysteine concentration in the induced sample.

To address this problem we have analyzed the toxicity effect of CysE production by counting the number of colonies survived after 8 hours.

Figure 5: Toxicity effect of CysE by serial dilution. NEB10β cells transformed with Part BBa_K731030 were grown in MOPS supplemented with glycerol (60 mM, MOPS A) and glucose (30 mM, MOPS B). After 4 and 8 h of induction with arabinose a 500 μl aliquot of cells was taken to make serial dilution on LB agar plates. Cell number was calculated with the following formula: Cells/mL = (# colonies counted)*(dilution factor)/(mL of culture plated).

The serial dilution test confirmed our theory: when cells are induced in glycerol they show very high expression levels, but few cells can cope with this pressure. Even if each of these few cells is actually producing much CysE, the cell doesn’t have the capabilities to sustain such high cysteine production, which is not achieved in a single step reaction (i.e. a bottleneck is reached with other enzymes).
When cells in glycerol are not induced, CysE production is decreased, allowing more cells to survive and producing higher amounts of cysteine.
In glucose the situation is the opposite: we have many cells, but low expression due to the glucose inhibitory effect on the araCp-BAD promoter. All these cells are producing low amounts of CysE, leaving the cells more “healthy” and able to produce high cysteine concentrations.

To summarize we decided that MOPS supplemented with glucose was the best compromise between protein expression and cysteine production, which ultimately means more sulfate reduced!

We assessed the production of H₂S by several qualitative and more quantitative assays:

• Gas Chromatography
• Lead acetate strips
• Copper precipitation
• Triple sugar iron test

Rotten eggs are dangerous?

Up until now we used colorimetric assays and standard curves to prove and quantify the presence of H₂S. However, hydrogen sulfide is a (toxic and poisonous) gas and as such it can be detected by Gas chromatography. To confirm the presence and estimate the concentration of H₂S produced we asked the help of an expert in gas Chromatography in the physics department. Damiano Avi came one day to the lab with his portable gas chromatographer (MICROGC A3000 Agilent) equipped with a 50C Poraplot U column for the detection of H₂S. Damiano keeps his H₂S stock in 10 L champagne bottles, because they can hold really high pressure!

Figure 8: PANEL A: 50 mL of cells were grown in LB in a 250 mL sterile bottle with a modified screw cap that allows to connect the bottle directly to the instrument. PANEL B: After 4 hours of induction, with 0.1 mM IPTG, the bottle was attached to a portable gas chromatographer (MICROGC A3000 Agilent). PANEL C: H₂S formation was qualitatively assessed by exposure to lead acetate test strips for few seconds. PANEL D: Gas Chromatography analysis of NEB10β cells with and without BBa_K731480. Measurements were taken 3 times at intervals of 2 minutes. A calibration curve was done with H₂S.

We estimated that a sealed 50 mL culture of E. coli carrying our BBa_K731480 after 4 hours of induction accumulated between 20 and 30 ppm of H₂S in a 250 mL bottle. This of course is the concentration of H₂S accumulated in the sealed bottle. If the bottle were open, such a high value would not have been reached.
As you can read in our H₂S section these concentrations are not an immediate risk for human health. Nevertheless is absolutely not advisable to inhale (or smell) such quantities for a prolonged time, and for this reason we took all the possible precautions to work in the safest conditions (see more in our safety section).

Measuring H₂S production by an indirect method: free copper concentration in the solution

In this second approach we exploited the formation of complexes between sulfide and metal ions. Briefly, adding a copper salt to the medium of an induced culture we observed a decrease of the free copper concentration as function of time, due to the precipitation of copper sulfide. The free copper concentration was measured after adding bathocuproinedisulfonic acid (BCS) (a chemical which turns orange when it reacts with Cu²⁺ and measuring the absorbance by UV-VIS at 483nm. We tried to obtain an estimation of the free copper using a standard curve made with different concentrations of Cu²⁺. This test was done using only once using Part BBa_K731400 and therefore quantitative data cannot be extrapolated surely.

Figure 9: Copper precipitation by CysDes. Cells transformed with Part BBa_K731400 (i.e. CysDes) were grown in LB in the presence of 2 mM Copper sulfate.To the supernatant it was added 1 µl of a 100 mM solution of Bathocuproinedisulfonic (BCS) reagent and 1 µl of a 1 M ascorbate. Absorbance was measured at 483 nm. Panel A: Uninduced cells, Panel B: Induced cells. Absorbance at 483 nm is shown in blue, optical density is shown in red. Read more details about this test in our protocol page.

More metals to be precipitated: the TSI plates

We learnt that sulfide reacting with metal ions produces a change in color. In this test we grew cells in triple sugar iron medium and observed the formation of black spots due to the precipitation of iron sulfide. The process was a bit slow, but after a week we observed clearly the “H₂S” formation, suggesting that when lacking oxygen our CysDes producing bacteria do not work as efficiently, as in aerobic conditions.

Figure 10: Cells transformed with BBa_K731480 were induced with 0.1 mM IPTG and a 5 µl aliquot was placed inside the iron containing medium with a tip. Cells were left at 37°C for 1 week. After 48 hours the first black spots started appearing. The maximum production of FeS was reached after 1 week.

Cotransformation

In order to be able to co-express CysE and CysDes we subcloned Parts BBa_K731030, BBa_K731400 and BBa_K731480 in the low copy vectors pSB3C5 and pSB4K5.

We tested protein expression levels of CysE upon induction of CysDes by measuring fluorescence intensity of CysE-sfGFP (i.e. BBa_K731040) produced when CysDes (BBa_K731400) was uninduced or induced with IPTG.

Figure 14: NEB10β cells cotransformed with Parts BBa_K731040 and BBa_K731400 were grown in LB and induced with 5 mM arabinose and different concentrations of IPTG. Time points were taken at 4 hours and 8 hours of expression to measure fluorescence intensity, as an indication of CysE expression levels.

The data show that CysE induction seems to be decreased significantly upon CysDes expression. For example, at 0.02 mM of IPTG the expression seems to be 3 fold less when compared with the levels of protein produced in the absence of IPTG.

To optimize CysE production we tried to induce the expression of the two proteins at different times. In the figure below it is shown the production of CysE, measured by fluorescence intensity, when CysDes was induced 4 hours later with 0.1 mM of IPTG.

Figure 15: NEB10β cells co-transformed with Parts BBa_K731040 and BBa_K731400 were grown in LB and induced with 5 mM arabinose when it was reached an OD of 0.6 (time zero) and with 0.1 mM of IPTG 4 hours later. Time points were taken every hour to measure fluorescence intensity, as an indication of CysE expression levels.

The data showed that we reached a good compromise in CysE expression levels over time when delaying the induction of CysDes.

Based on our findings we decided that the conditions to be used on future applications on the statues would be:

• MOPS supplemented with 30 mM of glucose
• expression of CysE with 5 mM arabinose
• expression of CysDes with 0.1 mM of IPTG after 4 hours from the induction with arabinose.

Co-culture

Unfortunately we were not able to look in the co-transformed cells at the expression of both proteins because we lacked fluorescent tagged parts with antibiotic compatibility.

Therefore we monitored CysE and CysDes expression levels over time in co-cultured NEB10β.

We established the following co-culture tests:

A) CysE-sfGFP + CysDes using Parts BBa_K731040 and BBa_K731400.
B) CysE + CysDes-sfGFP using Parts BBa_K731030 and BBa_K731480.

For co-culture A we used the following growth conditions:

+5 mM arabinose, - 0.1 mM IPTG
+5 mM arabinose, + 0.1 mM IPTG

For co-culture B we used the following growth conditions:

–5 mM arabinose, + 0.1 mM IPTG
+5 mM arabinose, + 0.1 mM IPTG

and checked fluorescence intensity of the sfGFP tagged protein being used as reporter. For example, we were able to look at CysE expression levels when CysDes was uninduced or induced with IPTG (co-culture A) and viceversa (co-culture B).

Figure 16: Top Panel CysE-sfGFP levels monitored by fluorescence intensity levels when CysDes is uninduced (+Arabinose -IPTG) and induced (+Arabinose +IPTG). Bottom Panel: CysDes-sfGFP monitored by fluorescence intensity levels when CysE was uninduced (-Arabinose +IPTG) and induced (+Arabinose +IPTG).

The data showed that induction of both CysE and CysDes had a synergistic effect on the expression levels of both enzymes. However, this is in contrast with what we saw with the co-transformed cells. This is possibly due to a much higher energetical request of the cell when the two enzymes are expressed simultaneously. This theory finds support on the fact the optical density levels recorded were significantly decreased when both genes were induced (about 30% less, data not shown).