Fig. 3: Flow cytometry measurement data of JeT_CMV (REU) in different cell lines. The three cell lines MCF-7, U2-OS and HeLa were cotransfected with the JeT_CMV promoter coupled to GFP and a reference plasmid including the promoter JeT coupled to mCherry. The relative fluorescence (REU) of GFP was measured 20 hours after transfection. The MCF-7 and the U2-OS cell line were measured four times and the HeLa cell line five times. The standard error of the mean (SEM) is indicated by the error bars.

As a first application of RA-PCR, we have created a library of constitutive promoters. We performed RA-PCR on oligos containing binding sites for some well known generally activating transcription factors (Sp1, Ap1,CREB, NF-Y) which we identified from literature search [2],[3]]. We also added NF-κB responsive oligos as NF-κB has non-specific activity and is therefore used by a variety of viral constitutive promoters, e.g. the HIV promoter [4]. We picked 24 colonies, two of which we dismissed after a test digest (not shown). Fig. 5 shows the sequence analysis of some randomly selected clones and demonstrates that RA-PCR is able to generate randomized repeats of oligos. We then measured the activity of the clones we picked by applying the Concept of Relative Expression Units (REU) we developed. Fig. 4 shows that we have been able to create a library of promoters of varying strength, some of which have an expression strength higher than JeT (which was not accomplished by JeT's developers, although attempted [1]). Such a library is of great value for fine-tuning gene expression levels. Selected clones were characterized by an independent method (image analysis) and submitted to the registry.

Fig. 4: A library of constitutive promoters created by RA-PCR Promoters were analyzed by the standards developed in the Measurement part of our project in HeLa cells.

Fig. 5: RA-PCR generates randomized repeats of transcription factor binding sites. Sequence analysis of clones of constitutive promoters generated by RA-PCR. Transcription factor binding sites are marked in color, random sequences in light grey. Annealing sequences are shown in bold and bold italic.

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203119 BBa_K203119]

We characterized clone 31 of our library of NF-κB responsive promoters by flow cytometry (3 independent experiments, each experiment having 3 replicates) and found it to be upregulated by approx. 100% (Fig. 6) in response to TNF-α. We also did a timecourse measurement of induction (Fig. 7). To verify our assumption that promoters created by RA-PCR are specific to the pathway of interest, we tried to induce clone 31 by a wide variety of conditions (full medium, full medium lacking growth factors / serum, minimal medium / starvation and pPARγ induction by Thiazolidinedione) and found no change in indcution levels (Fig. 8). Also, we show that the promoter is active only if p65, a component of NF-κB, localizes to the nucleus (Fig. 9).

Fig. 6: BBa_K203119 is a promoter induced approximately by 100% by TNF-α. REU was determined by flow cytometry, 3 experiments each with 3 replicates	Fig. 7: Timecourse of induction. Measured by image analysis. See discussion for explanation of the deviation between the two methods.
Fig. 8: BBa_K203119 responds only to TNF-α and is not induced by other conditions. U2-OS Cells transfected with BBa_K203119 in BBa_K203100 were subjected to a variety of conditions (full medium DMEM+++, full medium lacking growth factors / serum DMEM++, starvation by minimal medium and pPARγ induction by Thiazolidinedione) and did not show any significant induction or repression.	Fig. 9: BBa_K203119 is only active if NF-κB is localized to the nucleus. Cells were cotransfected with BBa_K203119 in [http://partsregistry.org/Part:BBa_K203100 BBa_K203100] and a p65-mCherry-fusion protein (kind gift of N. Brady, BioQuant). Only if the red fluorescence is localized to the nucleus, bright green fluorescence can be observed.

Targeting Signals

Main article: Outlook and Summary

Protein domains for targeting to sub-cellular compartments. We intend to use this to visualize multiple pathways. All parts below were characterized in devices consisting of [http://partsregistry.org/Part:BBa_I712004 BBa_I712004 (CMV)], the targeting signal, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203103 BBa_K203103 (GFP)] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203102 BBa_K203102 (PolyA signal/terminator)].

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203000 BBa_K203000]

Shown to localize proteins to the plasma membrane.

[http://partsregistry.org/Part:BBa_K203101 BBa_K203101]

Shown to localize proteins to the ER membrane. An expression device consisting of Part:BBa_I712004 (CMV) Part:BBa_K203103 (GFP), Part:BBa_K203101 (Sar-1) and Part:BBa_K203102 (terminator) was assembled for this characterization.

References

[1] Tornoe J. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 297: 21-32 (2002).

[2] Edelman G. M., Meech R., Owens G. C. & Jones F. S. Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. PNAS 97: 3038-43 (2000).

[3] Ogawa R. Construction of strong mammalian promoters by random cis-acting element elongation. Biotechniques 42: 628-632 (2007).

[4] Rattner A. NF-κB activates the HIV promoter in neurons. EMBO 12: 4261–4267 (1993).