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Part:BBa_K082034:Experience
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Characterization of BBa_K082034 by ETH Zurich 2010 iGEM Team
Introduction
The iGEM 2010 Team of ETH Zurich considered using this part as a reporter system to evaluate the [http://2010.igem.org/Team:ETHZ_Basel/Biology/Cloning cloning strategy]. Since the part contains a LacI binding site on the operator the expression behaviour under different concentrations of LacI and binding sites were examined.
Plasmids
| plasmid | purpose | origin | resistance | additional information |
|---|---|---|---|---|
| pSEVA132 | expression of BBa_K082034 | pBBR1; approx. 75 copies/cell | kan | Victor de Lorenzo's lab; analysis of copy number (pSEVA132 = wv1) |
| pKQV4 | expression of lacI | pBR322; high copy | tet, amp | [1] |
Cloning
pSB1A2_BBa_K082034 and pSEVA132 were digested according to the protocol found [http://openwetware.org/wiki/Engineering_BioBrick_vectors_from_BioBrick_parts/Restriction_digest here]. The restriction enzymes used were EcoRI and PstI. The part BBa_K082034 was then isolated from pSB1A2 with an agarose gel and ligated into pSEVA132 according to the [http://www.neb.com/nebecomm/products/protocol2.asp quick ligation protocol] of New England Biolabs to give rise to the plasmid pSEVA132_BBa_K082034.
control digest of pSB1A2. lane 1: [http://www.neb.com/nebecomm/products/productn0468.asp 1kb ladder]; lane 2: digested pSB1A2, part at 1.1kb, vector at 2kb.
control digest of pSEVA132. lane 1 and 6: [http://www.neb.com/nebecomm/products/productn0468.asp 1kb ladder]; lane 2 and 4: pSEVA132_BBa_K082034 not digested; lane 3 and 5: digested pSEVA132_BBa_K082034, part at 1.1kb, vector at 4.5kb.
Expression Behaviour of BBa_K082034 in pSEVA132
Methods
From an initial culture of E. coli DH5α cells (5 ml LB in 15 ml Falcon tube, incubation overnight at 37°C, 220 rpm) containing either only pSEVA132_BBa_K082034 or pSEVA132_BBa_K082034 and pKQV4_lacIq, cultures (10 ml LB in 100 ml Erlenmayer flask) were inoculated to an OD (at 600 nm, measured with Eppendorf Biophotometer, path length 1 cm) of 0.05. Fluorescence (excitation at 485 nm and emission at 530 nm) and optical density at 595 nm were measured in a microtiterplate contatining 200 μl of samplte with a PerkinElmer Victor3 Fluorometer at time intervals of 15 min. After 1 hour of incubation (37°C, 220 rpm) expression was initiated by 1 mM IPTG. The obtained values for fluorescence and optical density were corrected by the values of an LB blank.
Results
| pSEVA132_BBa_K082034, induction at 60 min. | ||
| pSEVA132_BBa_K082034, no induction. | ||
| pSEVA132_BBa_K082034 and pKQV4_lacIq, induction at 60 min. | ||
Conclusion
Cells harboring pSEVA132_BBa_K082034 showed an increase in fluorescence when induced with IPTG. However, if not induced, some leaky expression of BBa_K082034 could still be observed. In contrary, cells harboring pSEVA132_BBa_K082034 and pKQV4_lacIq did not show any increase in fluorescence, even at an inducer concentration of 1 mM. The increased level of LacI provoked by pKQV4 seem to shut off GFP production completely. Cells harboring pSEVA132_BBa_K082034 seem to produce enough LacI in order to repress the load of BBa_K082034 brought to them, at least to some extent.
If introduced into a medium to high copy plasmid, BBa_K082034 may only be useful to determine its presence/absence. However, if a tightly regulated expression of BBa_K082034 is required levels of BBa_K082034 and of LacI would need to be carefully adjusted. Too much BBa_K082034 leads to leaky expression, as seen with the medium copy plasmid pSEVA132_BBa_K082034. Too much LacI, on the other hand, might completely block expression even if induced, as seen with pSEVA132_BBa_K082034 in combination with pKQV4_lacIq.
Reference
[1] [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC400994/pdf/emboj00129-0314.pdf Strauch, M. A.; Spiegelman, G. B.; Perego, M.; Johnson, W. C.; Burbulys, D.; Hoch, J. A. The transition state transcription regulator abrB of Bacillus subtilis is a DNA binding protein. EMBO J. 1989, 8, 1615-1621.]
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