Revision as of 19:14, 26 September 2012

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Last year's project: Synchronized Oscillatory System

Wageningen UR

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Parts

In the Open Access scientific community that iGEM is, the Registry of Standard Parts plays a crucial role. As teams construct new parts and devices, they make them available to anyone who might want to use or improve them. However, the current registry can be hard to navigate (see The Constructor) and turns out to contain non-functioning and even non-existent parts. To work towards a better Open Source future, we think it is of great importance that apart from quantity, the quality of the registry should improve as well. It is up to all users to make this happen. We tried to contribute to the quality of the registry not only by sending in our bricks (standardized tools - they were all sequenced), but also by validating other bricks and improving some.

Parts we made

<groupparts>iGEM012 Wageningen_UR</groupparts>

Parts we used

For our project, we used few bricks from the 2012 Spring Distribution Kit. A list is included below.

Part	Name	Description
[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]	Terminator	double terminator (B0010-B0012)
[http://partsregistry.org/Part:BBa_I13522 BBa_I13522]	pTet GFP	Untagged GFP behind a constitutive promoter.
[http://partsregistry.org/Part:BBa_J04450 BBa_J04450]	RFP Coding Device	RFP Coding Device (iptg + Cap induced, pSB1C3 backbone)
[http://partsregistry.org/Part:BBa_J04500 BBa_J04500]	IPTG inducable promotor with RBS	R0010.B0034
[http://partsregistry.org/Part:BBa_K197038 BBa_K197038]	BBb - BseRI/AcuI entry vector	This part is and entry vector in BBb Format. It is flanked by BamHI, BseRI, BglII, and AcuI sites and is used in the 2AB Layered Assembly scheme developed at UC Berkeley.

Parts we ordered

For our project, we ordered few bricks from the iGEM Registry. A list is included below.

Part	Name	Description
[http://partsregistry.org/Part:BBa_K197021 BBa_K197021]	EILD	One peptide of a heterodimeric leucine zipper that dimerizes with KILR.
[http://partsregistry.org/Part:BBa_K197022 BBa_K197022]	KILR	A heterodimeric peptide that dimerizes to EILD. This can be used for adhering two cells together to evolve new two cell chemistry and functions.
[http://partsregistry.org/Part:BBa_J3901 BBa_J3901]	PrFe-mRFP1	This device coupled the promoters PI and PII of rus operon from Acidithiobacillus ferrooxidans with a monomeric red fluorescent protein (BBa_E1010) used as a signal, showing specific sensorbility response to iron ions presence.
[http://partsregistry.org/Part:BBa_I765010 BBa_I765010]	Iron promoter -EYFP reporter	Iron promoter expresion is reported by the trascription of EYFP reporter.

Parts we standardized

Two of these parts ([http://partsregistry.org/Part:BBa_K197021 BBa_K197021] and [http://partsregistry.org/Part:BBa_K197022 BBa_K197022]) were in a non-standard backbone and format ([http://partsregistry.org/Part:BBa_K197038 BBa_K197038]). For the [http://partsregistry.org/Part:BBa_K197022 BBa_K197022] part, we tried to brick it by adding standard 10 pre- and suffixes and inserting it in the pSB1C3 (we used [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] as template) standard backbone. We uploaded the standardized version of [http://partsregistry.org/Part:BBa_K197022 BBa_K197022] on the registry as [http://partsregistry.org/wiki/index.php?title=Part:BBa_K883600 BBa_K883600]. Unfortunately the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K883600 BBa_K883600] lacks a SpeI restriction-site to match iGEM's standard 10. We found out about this after the part was send to the parts registry. It will be corrected by a new ligation in a working part after the wiki freeze.

Faulty parts

Table 1

Table 2

Table 3

Even though the Registry constitutes the main repository for the iGEM competition, it contains a large number of parts that have a questionable status. Only 20% of all biobricks have been validated (Table 1). This relates to 4.083 validated parts in a registry of 19.976 in total. A mere 5% of all biobricks have been sequenced (table 2) and only 35% of all biobricks are available (table 3). Data was collected from The Constructor Database. Users of biobricks have the responsibility to sequence used constructs and assess their validity, which they can describe in the experience tab of the registry page. Such initiatives improve the quality of the Registry and benefits all users.

In our project, we aimed to load VLPs with iron, after which the iron could be detected using brick [http://partsregistry.org/Part:BBa_J3901 BBa_J3901] , which is an iron reporter. And so we ordered the brick and started experimenting with it. When grown on LB agar, the cells showed to express RFP constitutively (See figure x). We imagined that LB might contain iron in such high quantities, that the threshold of the iron inducable promotor was exceeded. In other words, the promotor would be extremely sensitive to iron. It was decided to make iron-free medium. Unluckily, the cells did not grow on this medium. In the meantime, this construct was sent for sequencing. Once we received the results, it showed that the biobrick was actually completely lacking its iron inducable promotor.
Another biobrick, [http://partsregistry.org/Part:BBa_I765010 BBa_I765010], was then ordered. This iron reporter supposedly had an iron inducable promotor in front of a YFP gene. After receiving sequencing results for this construct, it became clear that this biobrick was also lacking its promotor.

We documented our findings extensively on the experience page of both parts [[http://partsregistry.org/Part:BBa_J3901:Experience 1], [http://partsregistry.org/Part:BBa_I765010:Experience 2]].

Students should be encouraged to validate existing biobricks and document their results in a good manner. It is nice to produce new and original biobricks, but it is also of utmost importance to validate the biobricks already present in the parts registry.

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