Part:BBa_K2602020

VHb under strong Anderson promoter + GFP under strong Anderson promoter

This is a VHb under an anderson promoter (relative strength 100%) + GFP under an anderson promotor (relative strength 70%)

Short description:
Co-expression of Vitreoscilla hemoglobin under an Anderson promoter of relative strength 16 % different strengths and GFP under an Anderson promoter with medium-high strength level.

Introduction
This composite part collection contains the Vitreoscilla hemoglobin (VHb) and the gene that codifies the green fluorescent protein (GFP). The coding sequences are controlled under two different Anderson promoters with their correspondent ribosome binding sites and a double terminator sequence to allow for a tight expression of each gene. The parts were used by Team Lund 2018 to test Vitreoscilla hemoglobin expression levels for protein co-expression.

This set of composite parts was created by Team Lund 2018 to allow for a modulated expression of the Vitreoscilla hemoglobin. This allows the user to screen various expression levels of the protein so that a suitable effect on the cell productivity is obtained when expressed under low oxygen environments. Co-expressing of hemoglobin with a target protein of interest to increase yields in e.g. reactor settings or to increase cell survivability under stressful environments [1].

Experience
All the parts were confirmed with agarose gel electrophoresis (fig. 1). NotI restriction sites are located in both the prefix and the suffix of the inserts, so there should be one band at 1563 bp corresponding to the VHb-GFP construct, and one at ~2200 bp for the plasmid backbone. In the figure it can be clearly seen that all the constructs, except K2602022 (no successful transformation), show the two bands expected from digestion with NotI.

Figure 1: Agarose gel electrophoresis results of the VHb-GFP constructs digested with NotI. From left to right: ladder, K2602020-K2602026 except K2602022. Marked bands on the ladder are approx. 1500 and 2500 bp.

After successful transformations, we performed SDS-PAGE (fig. 2). From the second column from the left to right it is possible to observe the protein profile of the constructs with the following BioBricks: BBa_K2602020, BBa_K2602021, BBa_K2602023, BBa_K2602024, BBa_K2602025 and BBa_K2602026. VHb has a molecular weight of 15.8 kDa which is expected to be just above the second band of the ladder, while GFP has a molecular weight of 27 kDa which is expected to be just above the fourth band of the ladder. The gel shows that all our transformations have the bands with the expected molecular weight.

Figure 2: SDS-PAGE results. From left to right: ladder, K2602020-K2602026 except K2602022

It is important to highlight that no negative control was used, as our control strain also has a band with a molecular weight like one of VHb and therefore the confirmation of the expression of VHb should be done by its expression with a His-tag, which would allow for purification before performing the SDS-PAGE.

The expression of the VHb-GFP constructs confirmed that all the transformants were able to produce GFP as can be seen based on the characteristic green color of the cell pellets (fig. 3).

Figure 3: From left to right with two tubes each: BBa_K2602023, K2602020, K2602021, K2602024, K2602026 expressed in <i>E. coli BL21. </i>

Absorbance of was GFP measured at 504 nm for the different promoter strengths, see fig. 4 . The graphs show that the higher the promoter strength, the higher the absorbance, i.e. concentration, of GFP.

Figure 4: Absorbance measurements of GFP for different promoter strengths of VHb.

Source The subpart expressing the hemoglobin is K2602010, with the relative promoter strength x.x, and the part expressing the GFP is BBa_J364000.

References
[1] Geckil H, Gencer S, Kahraman H, Erenler SO (2003) Genetic engineering of Enterobacter aerogenes with the Vitreoscilla hemoglobin gene: cell growth, survival, and antioxidant enzyme status under oxidative stress. Res Microbiol 154:425–431

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
  Illegal NheI site found at 620
  Illegal NheI site found at 643
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1318