Part:BBa_K3144013
constitutive promoter + RBS + GST + Bacterioferritin + ter
This part is a composite part of bacterioferritin protein coding sequence, under control of constitutive family member, and strong RBS binding site and finally a double terminator to grant high expression rate in the cell-free kit system
Usage and Biology
In Silico Modeling
We thought of improving Bacterioferritin to be more stable as its predicted stability index is more than 40. Also increasing its solubility to make GRAVY more negative value, therefore we added GST to the protein sequence and re-modeled the protein and identified its stability index, GRAVY, and deltaG at room temperature. Using primary structure , we tested the solubility indicated by GRAVY and stability indicated by Instability Index to find that the addition of Gst reduced both of them as primary indicator
| Extiction Coeficcient | Gravy | PI | Instability Index | Aliphatic Index |
|---|---|---|---|---|
| 64540 | -0.471 | 5.47 | 41.92 | 90.97 |
A second indicator of the stability improvement upon GST addition was the thermostability curve, detlaG of transition at room temperature is an indicator to the stability ,while thermal resistance sometimes indicate the general stability. Often increasing thermal stability could be met by three strategies one of which is heat capacity of transition becomes less negative, which leads to an increase of Tm through a modification of the shape of the curve* , upon producing the thermostability curve we found that the heat capacity become less negative and deltaG of transition increase in the negativity, curve indicated in the following figure:
https://2019.igem.org/wiki/images/9/9c/T--CU--Bacterioferritin_Stability.png
From in silico protein modeling results,we have chosen to tag bacterioferritin with GST tag and test the results in vitro, specially the stability before and after adding GST tag at room temperature as deltaG became more negative upon fusing protein in silico prediction
Experiments
Testing the bacterioferritin improvement after adding GST Tag
According to the modelling we performed, we combined GST and Bacterioferritin in one plasmid under the expression of tac promoter considering the folding time of the fused protein, then we tested the reduction of the TDS value of NaCl solution at room temperature, because bacterioferritin didn’t reduce TDS at room temperature unlike in 42 degree. From the results we found a great improvement in the ability of the tagged bacterioferritin to reduce the TDS of NaCl solution at room temperature, around 500 ppm, enabling the fusion protein to behave better in further application of the system
https://2019.igem.org/wiki/images/thumb/8/8a/T--CU--tag.gif/800px-T--CU--tag.gif.png
Sequence and Features| None |