Basic

Name	Type	Designer	Length
BBa_R0040	pTet promoter	June Rhee, Connie Tao, Ty Thomson, Louis Waldman Group: Antiquity (2003-01-31)	54 bp

Composite

Name	Type	Designer	Length
BBa_K1352000	Ag43 under pBAD/araC promoter	June Rhee, Connie Tao, Ty Thomson, Louis Waldman Group: Antiquity (2003-01-31)	54 bp
BBa_I746908	sfGFP under pBAD/araC promoter	Ana-Maria Cujba, Martyna Sroka Group: iGEM14_Aberdeen_Scotland (2014-10-01)	4493 bp
BBa_K1072000	Lux based quorum sensing system	Yichong wang, Rui chen, Xihao liao and Junjie zhang Group: iGEM13_SCUT (2013-08-31)	2910 bp
BBa_K525998	This part consists of a T7 bacteriophage promoter followed by a very strong RBS – BBa_B0034	Anna Drong Group: iGEM11_Bielefeld-Germany (2011-09-13)	32 bp

Description

BBa_R0040

This part was used as a negative control for fluorescence measurements, since it is non-coding except for chloramphenicol resistance.

BBa_K1352000

Ag43 under pBAD/araC promoter. This part codes with the Ag43 pro-protein under the control of the pBAD/araC regulation system.

Briefly, Antigen 43 (Ag43) is a bacterial cell adhesion outer membrane protein, belonging to the auto-transporter family. It consists of two subunits – β, a trans-membrane β-barrel protein and α, a secretory protein that non-covalently binds to the β subunit and is responsible for the aggregation by forming α-α dimers. These two subunits come from the same peptide chain that is cleaved by an aspartate protease domain in the same pro-protein. Phenotypically, Ag43 expression leads to inter-cell aggregation.

We used BBa_K1352000 as PCR templates to make tagged variants of Ag43 (BBa_K2168000 and BBa_K2168001). For a excellent description of Ag43, we recommend visiting . BBa_I746908

This part codes for sfGFP under regulation of pBAD/araC system. We used this part to characterize the pBAD/araC regulation system, more specifically promoter activity repression by glucose, since this had paradoxically not been checked in the past 12 years. For more details about experiments and results, please click .

BBa_K1072000

Lux based quorum sensing system.

Quorum sensing systems are biological regulation circuits that allow gene regulation dependent on local cell-densities. A well-studied example is that in Aliivibrio fischeri lux quorum sensing system.

The system works as follows. luxI gene under the pLuxI promoter encodes for the LuxI AHL-synthetase enzyme that synthesizes AHL, non-polar signalling molecules that can diffuse out into the extracellular environment. Another protein encoded by this part, LuxR from the luxR gene under the pluxR promoter, binds to AHL and the LuxR-AHL complex homodimerizes further to give a (LuxR-AHL) dimer. This dimer acts as a transcriptional activator for the pluxI promoter, leading to a positive feedback loop for luxI-luxR expression and AHL synthesis.

Further, the dimerization of the LuxR-AHL complex leads to a positive Hill coefficient for the process leading to a switch-like behaviour with respect to the AHL concentration. This lead to a switch like behaviour with respect to cell-density since the AHL production rate is dependent on the local cell density.

Hence, any gene under the pluxI promoter would show a switching behaviour dependent on cell-density. We decided to check the same for BBa_K1072000, one for the few parts that has a complete quorum sensing system with a fluorescent reporter. So far, the characterization of this part has a microscopy image of green fluorescent cells; however, quorum sensing can established only by a growth curve. We have shown quorum sensing by this part experimentally. For experimental details, please click .

BBa_K525998

This part consists of a T7 bacteriophage promoter followed by a very strong RBS – BBa_B0034. This part was chosen by us for collaborating with the on their . The activity of this promoter seems to be inconclusive

Name	Type	Designer	Length
BBa_K2168000		Arunavo Chakraborty Group: iGEM16_IISc_Bangalore (2016-10-13)	4511 bp
BBa_K2168001		Arunavo Chakraborty Group: iGEM16_IISc_Bangalore (2016-10-13)	5258 bp

BBa_K2168000

This is a modification of BBa_K1352000, where Ag43 has been tagged on the N-terminal with a 6xHis tag for purification and quantification. This part was prepared by Gibson assembly, the details of which can be found . The location of the 6xHis tag would be the most critical since the functioning of N-terminal signal peptide needs to be maintained for Ag43 to remain phenotypically active.

BBa_K2168001

This is a modification of BBa_K1352000, where Ag43 has been tagged at the N-terminal with 6xHis tag and at the C-terminal with a sfGFP tag. This part was prepared by Gibson assembly, the details of which can be found here. In addition to the location of the 6xHis tag, the linker between the Ag43 and the sfGFP coding sequences needs to be chosen with some thought. The linker that we choose as a (GGGGS)2 amino acid sequence, which would be a flexible linker with intermediate hydrophobicity.
However, there is a reasonable possibility of this part being non-functional since the C-terminal amino acid for Type V auto-transporter family (of which Ag43 is a member) seems to be critical for integration of the protein on the outer-membrane. Since the C-terminal of Ag43-sfGFP would no longer be free, issues with aggregation due to BBa_K2168001 were expected.
Experimentally, we found the over-expressed Ag43-sfGFP chimera to be present in cellular interior based on epifluorescence microscopy images of induced cells.

Ag43 measurement systems

Name	Type	Designer	Length
BBa_K2168000		Arunavo Chakraborty Group: iGEM16_IISc_Bangalore (2016-10-13)	4511 bp
BBa_K2168001		sfGFP under pBAD/ara promoter	Arunavo Chakraborty Group: iGEM16_IISc_Bangalore (2016-10-13)	5258 bp

The parts are Ag43 chimeras with reporters to quantify Ag43 concentration. BBa_K2168001 has both a fluorescent tag and a 6xHis tag to independently estimate the concentration of Ag43 and check the agreement of both methods, whereas BBa_K2168000 has only a 6xHis tag, in case the fluorescent tag interferes with aggregation due to Ag43 in BBa_K2168001. The common theme is to estimate Ag43 concentration and correlate it with the degree of aggregation, thus determining the feasibility of replacing or supplementing centrifugation with Ag43 aggregation.

Native lux system:

Given our interest in protein-over-expression, we designed a quorum sensing system to allow for greater protein-over-expression without leaky expression. For this, we hypothesized that have bicistronic expression system for luxI and GFP, since it decreases the competition for LuxR-AHL dimer by the pluxI. So, we ordered the native lux system codon optimized for E coli from IDT, however, we received empty tubes from them.