Enhancement

E. coli odor itself is not that pleasant as well. We use RED to knockout the insole production gene tnaA (tryptophanase) to reduce the odor itself. Insole in shoes is not a wonderful home for microorganisms to grow. We use vitreoscilla hemoglobin (existing part K1321200) to enhance the grow ability of two E. coli strains and get the quantitative measurement result of the Biobrick.

VHb Enhancement

Overview

Goal: To enhance the ability to live in tough environment for engineered E. coli strains.

Achievement: Observational measurement and quantitative measurement finished. Result shows that in restricted environment (liquid culture volume related), VHb can accelerate the growth and improve protein production in early stage when oxygen supply is efficient.

Part: BBa_K1919500

Introduction

Vitreoscilla hemoglobin (VHb) was first studied in the 1960s and recognized as a hemoglobin in 1986. It was the first bacterial hemoglobin discovered. It transfers oxygen to its host under oxygen limitation. Yeast two-hybrid studies showing direct interactions of VHb with the terminal respiratory oxidases cytochrome bo and cytochrome d and VHb enhancement of oxygen uptake and ubiquinol oxidase activities of respiratory membranes all support this role. In conclusion, it enhances the ability for E. coli to utilize oxygen to grow. [1]

Now it has been used for the bioproduct synthesis in many species.

In iGEM14_Imperial project, VHb is used in Gluconacetobacter xylinus strain to promote the production of cellulose. SCU-China 2016 are expected to express this gene in E. coli to promote the engineered bacteria growth ability in relatively bad condition (Shoes).

Design

Firstly, we raised a simple theory called “Energy balance theory” in modeling prediction for VHb.

If energy gained by VHb synthesis is more than energy cost on VHb synthesis, E. coli grow better.

If energy gained by VHb synthesis is less than energy cost on VHb synthesis, E. coli grow worse.

We guess the most important factor for the balance theory is depending on the oxygen concentration in environment, then we started to design the experiment and use modeling to predict the final curve like this (Figure 1).

Figure 1. X-axis refers to oxygen concentration and Y-axis refers to cell division speed for K1919500 strain. In low oxygen concentration, energy gained by VHb synthesis is more than energy cost on VHb synthesis, division speed is high than control. However, when oxygen concentration is higher, energy gained by VHb synthesis is less than energy cost on VHb synthesis because oxgen concentration is high, there’s no need for VHb to accelerate the oxygen supply. The result fits one Chinese paper published [2]. More information, please visit modeling part.

Except observational measurement, we designed the quantitative measurement in different oxygen concentrations to test its ability to promote the growth and protein production.

We ligate biobricks K1321200 (J23106 + VHb + Terminal) and J04450 (J23100 + mRFP + Terminal) to construct the Biobrick K1919500 to test.

The K1919500 is transferred into BL21(DE3) to grow on LB agar plate to observe its morphology.

Then, we plan to use oxygen packs and device from MITSUBISHI GAS CHEMICAL COMPANY, INC. There are three kinds of oxygen pack to construct different oxygen concentrations in a special plastic box: Anaero Pack (~0% oxygen), MicroAero Pack (~8% oxygen), CO2 Pack (~16% Oxygen). We put 20ml LB in the box and put in oxygen pack to construct different oxygen concentrations for E. coli to grow.

We want to test the time for the liquid culture to attain the stable oxygen concentration by dissolved oxygen meter. We want to cultivate K1919500 strain in different oxygen concentration and test the OD600 and fluorescence intensity with time to measure the VHb ability to promote the growth and protein production.

Results

SDS-PAGE results shows that it express successfully in E.coli (Figure 1).

Figure 1

The morphology plate experiment shows that compared to J04450, K1919500 strain colony is distinctly bigger and redder (Figure 2).

Figure 2.

Then, we did the pre-experiment to find when the oxygen concentration in 20ml liquid culture can attain stable condition. The result shows that for Anaero Pack 45min at ~0.40mg/L, for MicroAero Pack 3h at ~2.8mg/L, for CO2 Pack 1.5h at ~4.4mg/L. In air, the oxygen concentration is ~5.8mg/L. (File is in Notebook Part)

Why we use 20mL is because the dissolved oxygen meter need certain liquid depth to test. We need more liquid to provide data for modeling.

Figure 3. Cultivation Device.

Because our experiment need to add same volume of E. coli in liquid culture firstly, we dilute bacteria liquid culture to test OD600 to check if it’s direct proportion relationship to make sure that we can dilute different bacteria liquid culture in same concentration. The result shows that it fits quite good (Figure 4).

Figure 4. OD600 and dilution factor relationship. It’s direct proportion relationship

We wonder if the time for the oxygen concentration balance affects the growth of E. coli at early stage. Because if the growth speed has big difference between J04450 and K1919500, at the real oxygen stable time, the bacteria concentration is not same. Then we add 2ul OD600=0.2 bacteria liquid culture to check what influence it can bring. The result shows that microplate reader can’t distinguish the difference. (File is in Notebook Part) So we can ignore it.

But at same time, we found one important thing:

If the bacteria can be seen in liquid culture (turbid), the oxygen concentration is near to 0mg/L in culture. The supply of oxygen may be dependent on how oxygen enter the liquid (surface of liquid culture and shaking speed).

Then we did the OD600 and fluorescence intensity test in AnAero Pack, MicroAero Pack, CO2 Pack and in air.

We construct the device like this. In box, there’s no lid, but for air culture, lid is involved in experiment system.

To our surprise, in 20ml/50ml OD600 experiment, all oxygen concentrations show same tendency: at first, the K1919500 is quicker than J04450, but in later stage, J04450 can grow more, but K1919500 enter the plateau phase (Figure 5).

Figure 5.

However, fluorescence result shows that fluorescence for K1919500 is much higher compared to J04450 in AnAero Pack, MicroAero Pack and CO2 Pack. It’s opposite to OD600 result: at first, the K1919500 is slower than J04450, but in later stage, K1919500 produce more mRFP and J04450 enter the plateau phase (Figure 6).

Figure 6.

Air condition result is not regular (Figure 7).

Figure 7.

We calculate Fluorescence intensity/OD600, the result shows that FI/single bacteria seems to produce higher mRFP. Air experiment data is irregular due to fluorescence intensity result irregular (Figure 8).

Figure 8.

And we did find that the result is not good as what we think especially in OD600 test. Then we tried to cultivate bacteria in 96 plate to test repeatly. The result is still the same tendency as 20ml/50ml test in OD600, FI, and FI/OD600 (Figure 9).

Figure 9.

To combine the finding mentioned above, we thought maybe in two experiment conditions, all E. coli are processing anaerobic respiration in fact. To verificate this, we give up obtaining exact oxygen concentration data for modeling to test 5ml/50ml culture system in air.

The result strongly shows that in early stage, K1919500 grow much quicker than J04450, especially compared to 20ml/50ml system and K1919500 always produce more mRFP. Though tendency is the same as before, but variation is significant. The result shows strong effect that VHb enhances the bacteria growth before plateau phase. So it shows a fact that VHb only promote the growth in aerobic respiration condition and VHb function is not strong as expected that it can catch oxygen that efficiently (That’s the reason why we started the experiment in 20ml/50ml system). It’s strongly affected by environment oxygen concentration. The reason why K1919500 carrying capacity K is lower than J04450, we think it’s due to that they are different strains and K1919500 carry more gene to express so their ability to utilize nutrition is different, and K is different (Figure 10).

Figure 10.

If we consider the anaerobic respiration, the growth for VHb in low oxygen concentration should be the slowest because VHb only costs energy and brings nothing, in middle oxygen concentration growth speed should be high and in high oxygen concentration growth speed should be lower than that in middle oxygen concentration. In the end, the OD600 at 5ml/50ml system experiment shows the result as our expected. However, FI result shows the opposite. We can’t explain it (Figure 11). But we think maybe if we consider growth and protein expression together, there are more evidence to explain. But we didn’t have more acute data to analyze.

And we improve our modeling result to fit the anaerobic respiration finding. The result is as follows and it’s consistent with our data and reference.(Figure 12)

The Energy Balance Theory is verificated primarily.

Figure 11.

Figure 12.

[1] Stark, B. C., K. R. Pagilla, and K. L. Dikshit. "Recent applications of Vitreoscilla hemoglobin technology in bioproduct synthesis and bioremediation. " Applied Microbiology & Biotechnology 99.4(2015):1627-36.