Team:SVCE CHENNAI/Parts
PARTS
Basic Parts
OUR BEST BASIC PARTS
1.RNA pHmeter - Wild scar (BBa_K2379002) :
The RNA pHmeter consists of the SraF gene that functions as a pH-sensitive RNA element which will induce gene expression under alkaline conditions (pH 8.4). This part is a variant of the RNA pHmeter wherein the natural scar native to the SraF gene is conserved.
2.RNA pHmeter – Biobrick scar (BBa_K2379003):
This part is yet another variant of the RNA pHmeter wherein the natural scar is replaced with the conventional 6bp biobrick scar.The RNA pHmeter we provided had a 4bp natural scar region. Since the biobrick scar site has 6bp, we had issues in adapting it to the biobrick standard. To overcome this discrepancy, we worked on the effect of mutations in the RNA pHmeter, RNA design principles and the effect of varying the distance between Shine Dalgarno Sequence and start codon. With this, we were successfully able to engineer the pH riboswitch with a biobrick scar site that similar is in both thermodynamic properties and secondary structure to the wild type. Despite the modifications made in it’s gene sequence, the functionality of this part remained the same and the new part was validated in the lab too. Both of these parts will serve as a novel and valuable addition to the iGEM registry since there are currently no parts available in the latter for the regulation of translation based upon pH. Thus we submit these parts for this year’s iGEM competition for the award of BEST NEW BASIC PART.
RNA pH Meter
WHAT IS SPECIAL ABOUT THIS PART ?
This part regulates translation based on a pH stimulus. Hence, gene expression can be controlled to a larger extent by using this riboregulator. And this is crucial for the synthetic biology community as well as iGEM which aims at improving the quality of research through standard biological parts.
OUR BASIC PARTS COLLECTION.
| PART NAME | PART NUMBER | DESCRIPTION | LENGTH |
|---|---|---|---|
| TnaC | BBa_K2379000 | A leader peptide which regulates the transcription of downstream genes. | 75 bp |
| rut+RBS | BBa_K2379001 | Part that regulates transcription termination and anti-termination | 214 bp |
| RNA pHmeter (wild type) | BBa_K2379002 | A pH sensitive RNA element with wild scar | 207 bp |
| RNA pHmeter (biobrick scar) | BBa_K2379003 | A pH sensitive RNA element with biobrick scar | 209 bp |
| B.subtilis RNA Thermometer | BBa_K2379004 | RNA thermometer optimized for Bacillis subtilis | 99 bp |
| RNA pHmeter (without scar) | BBa_K2379012 | A pH sensitive RNA element with without scar | 203 bp |
Composite Parts
OUR BEST COMPOSITE PARTS
1. ADAPTOR (TnaC + RUT) (BBa_K2379008):
An ideal genetic regulator that combines the best properties of both transcriptional and translational regulation. This part when placed downstream of any RNA element has the ability to convert it’s translational regulatory property into a transcriptional regulatory one.
Adaptor
1.A) RNA Thermometer + Adaptor + GFP (BBa_K2379010) :
This part is a modulation of the adaptor with a temperature- sensitive element (RNA Thermometer 4U ). By placing this RNA element upstream of the adaptor element ( TnaC + RUT) , it can be made to regulate transcription of the downstream genes at higher temperature( above 37ºC).
Adaptor with RNA Thermometer
1.B) RNA pHmeter+ Adaptor + GFP (BBa_K2379011):
This variation of the adaptor consists of the pH-responsive RNA element (RNA pHMETER) placed upstream of the TnaC and RUT site. By this way, the adaptor enables the RNA pHmeter to control transcription of downstream genes at alkaline pH (pH 8.4).
Adaptor with RNA pH Meter
This adaptor can be a powerful tool in the hands of any synthetic biologist who intends to convert the translational regulation of their RNA element into transcriptional regulation.This is because the adaptor possesses the ability to be easily engineered and as well regulate the expression of multiple genes under a single promoter. Hence we submit these parts to this year’s iGEM competition for the award of BEST COMPOSITE PART.
WHAT IS SPECIAL ABOUT THIS PART ?
- OPERON REGULATION: Can control the expression of multiple genes under the influence of a single promoter and an RBS.
- GENETIC LOGIC GATES REGULATION: Can be easily assembled into higher order function(Genetic logic gates and invivo computational devices)
- METABOLIC ENGINEERING: Can be used to measure the FCC ( flux control coefficients) for several genes encoding for different enzymes that can be placed under a single promoter.
OUR COMPOSITE PARTS COLLECTION.
| PART NAME | PART NUMBER | DESCRIPTION | LENGTH |
|---|---|---|---|
| RNA Thermometer + GFP | BBa_K2379005 | Part used for characterizing the RNA Thermometer | 988 bp |
| RNA pHmeter +GFP (wild scar) | BBa_K2379006 | Part used for characterizing the RNA pHmeter with wild scar | 1077 bp |
| RNA pHmeter +GFP (Biobrick scar) | BBa_K2379007 | Parts used for characterizing RNA pHmeter with biobrick scar | 1095 bp |
| Adaptor ( TnaC + rut ) | BBa_K2379008 | A genetic tool that converts translationalregulation of riboswitches intotranscriptional regulation | 297 bp |
| Constitutive RBS + Adaptor + GFP | BBa_K2379009 | Adaptor with a constitutive translational regulator | 1207 bp |
| RNA Thermometer + Adaptor + GFP | BBa_K2379010 | Adaptor with temperature sensitive translational regulator | 1291 bp |
| RNA pHmeter+ Adaptor + GFP | BBa_K2379011 | Adaptor with pHsensitive translational regulator | 1398 bp |
REFERENCES
- Konan, K.V. & Yanofsky, C. Rho-Dependent transcription termination in the tna Operon of Escherichia coli: Roles of the boxA Sequence and the rut Site.J. Bacteriol. 182(14), 3981–3988 (2000).
- Chang, C. L.,Lei Qi, Lucks J.B., Segall-Shapiro T.H., Wang, D.,Mutalik V.K.& Arkin, A.P.An adaptor from translational to transcriptional control enables predictable assembly of complex regulation. Nat. Methods 9,1088–1094(2012).
- Richardson J.P. Loading Rho to terminate transcription. Cell 114, 157–159 (2003).
- Nechooshtan, G., Elgrably-Weiss, M., Sheaffer, A.,Westhof, E., & Altuvia, S. A pH-responsive riboregulator.Genes Dev. 23, 2650–2662 (2009).
- Nechooshtan, G., Elgrably-Weiss, M., & Altuvia, S.Changes in transcriptional pausing modify the folding dynamics of the pH-responsive RNA element. Nucleic Acids research. 42(1), 622-630 (2013)
- Bingham, R., Karen, H., Joan S. Alkaline induction of a Novel Gene Locus, alx, in Escherichia coli. Journal of Bacteriology. 172(4), 2184-2186(1990)
