TARGET BANNER

Background

When a favorite protein (afp) is cloned into the target vector ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K215002 BBa_K215002]) two tags are fused onto the N and C terminal of afp. These tags are depicted below:

The first key feature of the target vector is the NheI restriction site, since this is where afp get's inserted. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the target vector at the NheI site (for detailed protocol see: NheI Insertion Protocol).

At the N-terminus of the Target is the display (aka Nano) tag, which is a 15 amino acid sequence that binds to streptavidin. Since streptavidin is being displayed on the surface of the cell this allows our protein to stick to the outside of the cell, but can still be released by the addition of biotin. For more details see: Cellular Surface Display System .

At the C-terminus of the Target is a secretion tag (prtB) that is recognized by the Type I secretion system, which secretes proteins from the cytosol, through the periplasim, and into the media. For more details go to: Secretion System .

Flanking each side of the NheI site are 6 consecutive hisitidines (6x-His) and TEV protease sites. The histidines allow for traditional immobilized metal affinity chromatography (IMAC) protein purification. The TEV sites allows for the N and C terminal tags to be cleaved off of afp, and due to the strategic placement of the 6x-His tags these tags can then be seperated from afp by simply running the cleaved solution over a column in which the tags stick but afp flows right through.

Experiments

For the target vector our goal was to:

• Construct the target vector
• Insert GFP into the target vector and characterize expression and function
• Biobrick and characterize OpdA, a nerve agent degrading enzyme
• Insert OpdA into the target vector and characterize expression and function

Target Vector Construction

To create the target vector we first synthesized a coding sequence that would produce the protein as described above. To do this we synthesized a gene from oligo's as described in our Gene Synthesis Protocol.

After creating the target construct, we created and BioBricked an expression vector (BBa_K215000) which would express the target protein upon induction with IPTG. We then added the target construct into the expression vector using standard assembly.

GFP Insertion and Characterization

Upon completion of constructing the target vector our first experiment was to determine if GFP was still functional as the fusion protein. To do this GFP (E0040) was inserted into the target construct use the NheI method as described above. After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was transformed into BL21(lacIq) cells, and subsequently grown in the presence or absence of IPTG. As a control an untagged E0040 was cloned into our expression vector and also grown in the presence or absence of IPTG. This would allow us to deteremine the effects of the tags on fluoresence. The cells were then washed with PBS, normalized to the same cell density, and fluoresence measured using an excition of 485 and emmision of 525 (cutoff at 515) in a XXXXX plate reader. The data is show below:

From this data we were able to conclude that our expression vector was functional, as is evident from the large increase in fluoresence with the addition of IPTG. We were also able to conclude that the Target-GFP is functional, but fluoresence was signficantly decreased.

In order to ensure that the Target-GFP had the appropriate 6x-his tags and that fluoresence was a function of protein concentration we purified Target-GFP using a traditional IMAC techniques (ADD TO PROTOCOLS). The protein concentration was measured from its absorbance a 280nm. A serial dilution of the protein was then made and the resulting fluoresence measured as described earlier. The data is shown below:

As expected the fluorescence intensity is linear with respect to protein concentration.

Protein 1

We inserted and characterized two proteins within the Target Vector: GFP (BBa_E0040) and Opda (BBa_K215090). (For more background information on the enzyme Opda, see here).

Whenever fusion proteins are created, it is never guaranteed that they will maintain their original solubility and function. Thus, after inserting a protein coding sequence into the target, it must be expressed and its properties re-characterized.

Protein 2

OpdA

OpdA (BBa_K215090) is an organophosphate-degrading enzyme from Agrobacterium radiobacter. It is capable of degrading a wide range of organophosphates, most notably pesticides that are poisonous to humans, such as paraoxon. We chose to biobrick and submit this enzyme to the registry for a number of reasons. First and foremost, this enzyme is easy to assay for since it can hydrolyze substrates very quickly (e.g. paraoxon) and form a bright yellow product. This yellow product would make it easy to see that the OpdA was present and functioning in our system. And secondly, OpdA is a very useful enzyme that could have applications in future iGEM and other synthetic biology projects, so its presence in the Standard Registry of Biological Parts is beneficial.

Experiments

To fully characterize this part, we expressed and purified the OpdA biobrick using traditional techniques. We then used this purified OpdA to generate the kinetic data shown below on the substrate paraoxon.

From the above plot, it obvious that this enzyme does not exhibit the usual Michaelis-Menten dynamics. It can be seen that at high enough concentrations, the enzyme actually undergoes substrate-inhibition, wherein the extra substrate actually slows the enzyme's velocity.

The plot below is zoomed in on the lower substrate concentration, where OpdA is not substrate inhibited.

The display/Nano tag was retrieved from Lamla and Erdmann: [http://www.ncbi.nlm.nih.gov/pubmed/14680960]

The secretion tag/prt B was retrieved from Palacios et al: [http://www.ncbi.nlm.nih.gov/pubmed/11157948]

The TEV Recognition Site, ENHLYFQG, was retrieved from [http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf]

Need citations and speak on literature!!!

After insertion of K215090 into the target construct, the tagged Opda (target-Opda) was found to be....

Insert target-Opda data here!!!