Charcterized parts

Measurement Kit

Main Article: Measurement

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203100 Part:BBa_K203100]

Fig. 3: Plasmid map of pSMB_MEASURE. BBB sites are shown in red, core promoter in light blue and proximal promoter in dark blue.

We separated JeT's core promoter from its proximal promoter by a HindIII site; it can therefore be used for screening of synthetic proximal promoters or for modifying the strength of a promoter by core promoter swapping. In addition, it contains a FRT site which will allow for stable integration into mammalian cells also containing a FRT site. Thus, it provides the possibility to characterize the promoter in a defined genome and in this way helps to avoid some of the challenges outlined above. For the same reason, it also contains a mammalian selection marker (hygromycine). For the generation of the plasmid, please see [http://partsregistry.org/Part:BBa_K203100:Design part design]. As a reporter gene, it contains GFP, which is followed by a SV40 mammalian terminator. We generated another plasmid pSMB_REFERENCE, which contains mcherry instead of GFP. It can be used for normalizations to transfection efficiency in Flow cytometry and image analysis.

Core promoters

Main Article: Core promoters

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203113 Part:BBa_K203113]

We cloned the CMV core promoter from [http://partsregistry.org/Part:BBa_I712004 Part:BBa_I712004] in front of the JeT proximal promoter from part [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203112 Part:BBa_K203112] to obtain JeT_CMV. We characterized this construct in three different cell lines, HeLa, MCF-7, and U2-OS and found it to have 50%-60% percent of JeT's activtiy depending on the cell line (Fig. 6). Thereby, the HeLa cell line was measured five times and the MCF-7 and U2-OS cell line measurements were performed four times. The fact that variations in the core promoter can be used to vary expression strength of a certain promoter of interest comes in useful if the transfer function of an existing promoter is to be altered, and it can be used to further diversify the synthetic promoters we created. We characterized these promoters by the same methods and accuracy as CMV and JeT/CMV (see Synthetic Promoter project)

Figure 6: 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.

Promoters created by RA-PCR

Main article: Synthetic promoters

All contain the core promoter of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203112 Part:BBa_K203112] and were screened / measured in [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203100 Part:BBa_K203100].

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203110 Part:BBa_K203110], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K203118 Part:BBa_K203118],[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203111 Part:BBa_K203111],[http://partsregistry.org/wiki/index.php?title=Part:BBa_K203109 Part:BBa_K203109] and others

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 [8],[7],[10]. 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 [13]. We picked 24 colonies, two of which we dismissed after a test digest (not shown). Figure 3 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. Figure 2 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 [10]). 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.

Figure 2: 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.

Figure 3: 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.

HEARTBEAT Predicted promoters

coming soon...

Natural promoters

Main article: Natural promoters

Targeting Signals

Main article: Outlook and Summary

Protein domains for targeting to sub-cellular compartments.