Difference between revisions of "Part:BBa K215002"

Latest revision as of 23:22, 18 October 2009

pLac+RBS+Secretion Signal and Streptavidin Binding Tags

K215002 drives the expression of BBa_K215001 by placing it under the control of a strong, IPTG-inducible promoter (R0011) and RBS (R0034). Any favorite protein (afp) can be inserted into a composite tag BBa_K215001. The composite tag has an NheI site (compatible with XbaI and SpeI, but make sure the resulting product stays in frame, described below) which places the afp in between a series of tags which can be used with the [http://2009.igem.org/Team:Washington University of Washington 2009 Ideal Protein Purification system (IPP)]. The series of tags are, respectively: a Nano-Tag (binds to streptavidin), a 6x His tag (for IMAC purification), a TEV cute site (for removal of N-terminal tags), the NheI site (insertion of afp), TEV, 6x His tag, and the 181 C-terminal amino acids of prtB (secretion tag recognized by the secretion system: BBa_K215107). In order for the secretion tag to signal the secretion of afp the BioBrick BBa_K215107 must be present as well.

Cutting a BioBrick coding sequence at X and S and pasting into the NheI site of this construct would result in a stop codon at the resulting scar site. Therefore, to insert a gene into the K215001 of this construct, a PCR product must be generated that keeps the gene of interest in frame with the rest of the sequence. To do this:

1. Design a primer that complements the coding sequence of interest starting at the second amino acid and includes the entire coding sequence except for the stop codon.
   1. Forward Primer: <8 random bp's> - <NheI or XbaI or SpeI> - <15-21bp complementing your gene of interest, starting at the second codon>
   2. Reverse Primer: <15-21bp complementing your gene of interest, ending BEFORE the stop codon (make sure it is a muliple of three to stay in frame)> - <NheI or XbaI or SpeI> - <8 random bp's>

1. Amplify the gene of interest
2. Clone into the NheI site of this construct.
3. Screen colonies with either the Forward Primer+VR or the VF2+Reverse primer for inserts in the appropriate direction.
4. Your fusion protein is tagged and ready for secretion and streptavidin binding!

Usage and Biology

To demonstrate the functionality of this construct, E0040 (GFP) was amplified as described above and inserted into the NheI site of BBa_K215002 and tested for expression. The constructs were then grown in BL21(lacIq) in the presence or absence of 0.5mM IPTG overnight at 298K. The two graphs below show the relative fluorescence of the two constructs as either a whole cell lysate, or a purfied soluble protein as a function of concentration. These data demonstrate that protein is produced from K215002, and (at least in the case of E0040) folds properly.

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  Whole Cell GFP Fluoresence To determine the effect of adding the N and C terminal tags to GFP, both BBa_K215000 (no N and C tags) and BBa_K215107 (N-terminal Nano-Tag and C-terminal PrtB Tag) with E0040 inserted were expressed in parallel. Cells were grown as described above. After harvesting, the cells were washed in PBS and tested for fluorescence using the parameters described above. Both constructs demonstrated strong fluorescence over background, though a significant decrease was observed for GFP after adding the N and C terminal tags.

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  Purified Tagged GFP Fluorescence To ensure the fluoresence observed in the whole cell lysate was due to soluble tagged GFP, tagged GFP was purified from cells, using a traditional protein purification technique described on [http://2009.igem.org/Team:Washington/Notebook University of Washington 2009 iGEM Notebook page]. The concentration of the purified protein was determined by measuring its absorbance at 280nm. The fluorescence (excitation 485nm, emission 525nm, cutoff 515nm on a SpectraMax M5e) was then measured as a function of protein concentration, and showed a linear correspondence.

Sequence and Features