Requirements

Bronze:

✔ 1. Team registration
Registered Teams

✔ 2. Complete Project Summary form
Our project is summarized on our wiki's front page and a preliminary description was sent to iGEM earlier in the summer.

✔ 3. Team Wiki
Berkeley's Wiki Home

✔ 4. Present a poster and a talk at the iGEM Jamboree

✔ 5. At least one new submitted and well-characterized standard BioBrick Part or Device or new application and outstanding documentation of a previously existing BioBrick part in the “Experience” section of that BioBrick’s Registry entry.

We submitted two new well-characterized BioBrick Parts:

1. BBa_K641008 is a general reporter for any ToxR system. When ToxR dimerizes it binds to Pctx and activates Transcription of genes downstream of Pctx. By putting ffGFP downstream of Pctx the strength of the ToxR construct dimerization is reported by a corresponding strength of GFP expression.

2. BBa_K641009 is a composite part that constitutively expresses a ToxR-LambdaRep construct. This ToxR construct will dimerize at a high affinity, due to Lambda Rep's dimerizing properties, making the cytoplasmic ToxR dimer an active transcription factor for the ctx Promoter. Thus, If this composite part is combined with a Pctx-GFP reporter there will be a high expression of GFP.

Silver (in addition to the Bronze requirements):

✔ 1. Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected

We submitted two new BioBrick Parts and characterized both on their respective Main Page. BBa_K641008, otherwise known as Bss52, was demonstrated to work as a reporter for the ToxR system. Bss52 is a composite part composed of the ctx Promoter and a coding gene for ffGFP. In the presence of a ToxR dimer Pctx will activate the transcription and thus the expression of GFP. The data can directly be seen here or on BBa-K641008's Main Page.

The second part, BBa_K641009, was demonstrated to function properly as a constitutive activator of the ctx promoter. This part is a composite part of a promoter, an rbs, and a ToxR chimera. This ToxR chimera is composed of a cytoplasmic ToxR and a periplasmic constitutive dimer that will cause two ToxR's to dimerize and activate Pctx. It activates Pctx at a very high level and thus when combined with BBa_K61008 will cause high expression of GFP. The data can directly be seen here or on BBa-K641009's Main Page.

✔ 2. Characterize the operation of at least one new BioBrick Part or Device and enter this information in the “Main Page” section of that Part’s/Device’s Registry entry.

The data for BBa_K641008 can directly be seen here or on BBa-K641008's Main Page.
In summary, the part was characterized by comparing the normalized fluorescence (RFU/O.D.) of several different constructs. There are 5 cell/plasmid combinations:
1. Plain MC1061 cells
2. MC1061 cells with the Bss52 Reporter by itself
3. MC1061 cells with a Constitutive GFP plasmid
4. MC1061 cells with two plasmids: a Bss52 Reporter and a ToxR-MukF variant that causes constitutive activation of Pctx
5. MC1061 cells with two plasmids: a Bss52 Reporter and a ToxR-MukF variant whose dimerization capabilities have been broken

As expected, Bss52 cells (2) were white in comparison to a constitutive GFP plasmid (3), and were not much brighter than plain MC1061 cells (1). Furthermore, the Pctx on Bss52 was shown to be inducible by a working ToxR chimera (4) showing an increase in fluorescence of a couple orders of magnitude. It was also shown that this activation is specific to a dimerizing ToxR since a ToxR-MukF whose dimerizing capabilities were broken by mutagenesis was unable to activate Bss52 and was just as white as plain Bss52 cells.

The data for BBa_K641009 can directly be seen here or on BBa-K641009's Main Page.
In summary, the part was characterized by comparing the normalized fluorescence (RFU/O.D.) of several different constructs. There are 4 cell/plasmid combinations:
1. Plain MC1061 cells
2. MC1061 cells with the Bss52 Reporter by itself
3. MC1061 cells with a Constitutive GFP plasmid
4. MC1061 cells with two plasmids: a Bss52 Reporter and a ToxR-LambdaRep producing plasmid

As expected, Bss52 cells (2) were white in comparison to a constitutive GFP plasmid (3), and were not much brighter than plain MC1061 cells (1). Most importantly, the Pctx on Bss52 was shown to be highly inducible by the LambdaRep ToxR chimera (4) showing an increase in fluorescence of a couple orders of magnitude.

Gold (in addition to the Bronze and Silver Medal requirements, any one or more of the following):

✔ 1. Improve an existing BioBrick Part or Device and enter this information in the Registry (in the “Experience” section of that BioBrick’s Registry entry).
With BBa_K641009, we are providing a functioning alternative to BBa_J07009 with characterization. The DNA in the Registry for the BBa_J07009 was inconsistent with the expected sequence, and no functional characterization was provided.

✔ 2. Help another iGEM team by, for example, characterizing a part, debugging a construct, or modeling or simulating their system
We worked closely with the Boston University/Wellesley Software team to evaluate and improve their Clotho software. Our team worked extensively with their latest build and dashboard, identifying bugs and making suggestions to improve the user experience. A list of our team's contributions to the BU/Wellesley effort can be found here.

☐​ 3. Outline and detail a new approach to an issue of Human Practice in synthetic biology as it relates to your project, such as safety, security, ethics, or ownership, sharing, and innovation.