Part:BBa_K1919500

K1321200+J04450 (Vitreoscilla hemoglobin + mRFP)

Vitreoscilla haemoglobin (VHb) can promote the cell growth and protein production ability. To measure the characters of Vitreoscilla haemoglobin generator constructed by iGEM14_Imperial (BBa_K1321200), we ligate mRFP generator (BBa_J04450) with VHb generator to test the protein production ability in E. coli.

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, for example). Except observational measurement, we did the quantitative measurement in different oxygen concentrations to test its ability.

In observational measurement, we use two E. coli BL21 strains which contain mRFP (BBa_J04450) and mRFP + VHb (K1919500) respectively to compare their growth and mRFP production by naked eyes. The result obviously shows that the colony is bigger and contains more mRFP.

Figure 1. Left is E. coli BL21 strain only containing mRFP. Right is E. coli BL21 strain containing mRFP plus VHb. The colony right is bigger and contains more mRFP.

In quantitative measurement, we use different oxygen concentrations (created by gas production pack) to test if VHb can really work. Because the synthesis of VHb cost energy. But if the volume of energy cost is lower than what VHb can bring, as a result, VHb makes the strain grow slower. We think oxygen concentration is an important factor. And we raise a theory called “VHb energy cost theory” in our modeling part. The result is shown in the part information page below, please visit our wiki for details http://2016.igem.org/Team:SCU-China/Model.The formation of mRFP needs oxygen molecule. So the fluoresence result remains considerations. But the OD600 data is reliable.

As the quantitative measurement results shown as follows, we conclude that in restricted environment, VHb can accelerate the growth and in early stage and it can improve the production protein. But all improvement occurs only when oxygen supply is efficient. For more details and analysis, please visit the wiki of SCU-China 2016 http://2016.igem.org/Team:SCU-China/Description.

Figure 2a. OD600 test shows in 20ml/50ml in different oxygen growth condition, the tendency is similar. It may be due to the high volume liquid culture in tubes and E. coli process anaerobic respiration.

Figure 2b. Fluorescence result shows the opposite tendency to Figure 2a. VHb + mRFP fluorescence is higher in later stage.

Figure 2b. Figure 2c. OD600/fluorescence result shows the same tendency as Figure 2b. Early data can be ignored because of the OD value in early stage. The results do show that single bacteria synthesizes more protein even in relatively anaerobic respiration condition.

Figure 3a. OD600 continuous test in 96 wells plate in air shows the similar result to 20ml/50ml test. It may be still due to the anaerobic respiration.

Figure 3b. Fluorescence result shows the same tendency as Figure 2b.

Figure 3c. OD600/fluorescence results shows the opposite tendency to Figure 2c.

Figure 4a. 5ml/50ml and 20ml/50ml cultivated in air OD600 test shows VHb does accelerate the growth of E. coli in early time for small volume cultivation. But at the end, because of the energy cost on VHb, strain containing mRFP + VHb has lower carrying capacity K.

Figure 4b. Fluorescence result shows the same tendency as Figure 2b. But the fluorescence is higher in 5ml system.

Figure 4c. OD600/fluorescence results shows the opposite tendency to Figure 4b. Early stage can be ignored.

Figure 5a. 5ml/50ml cultivated in different oxygen concentrations at 6h OD600 test shows VHb does accelerate the growth of E. coli. And the curve is matching with our VHb energy cost theory.

Figure 5b. Fluorescence result shows the opposite tendency as Figure 5a.

Figure 6. Modeling result for VHb protein function. The figure refers to the division of cell division cycle time of E. coli with VHb and E. coli without VHb. In low oxygen concentration, VHb strain processes anaerobic respiration and it only costs energy and brings nothing. VHb drags the E. coli growth and the division is negative number. As the oxygen concentration raises up, the VHb strain starts aerobic respiration and it utilizes oxygen better than strain without VHb. Division is positive number. When oxygen supply is totally content, the energy cost on synthesis VHb is less than what VHb can bring. The division is negative number. The result fits our experiment data and reference.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 477
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1293
  Illegal AgeI site found at 1405
  
• 1000
  COMPATIBLE WITH RFC[1000]