BBa_K1911002: ClpP
The ClpP gene codes for the ClpP subunit protein which works together with the ClpX subunit to break down proteins that are tagged with degradation tags. The ClpXP system identifies tagged proteins and proceeds to unfold the protein and break it down into individual amino acids. The ClpXP system along with degradation tags can be used to break down certain proteins which are produced by certain genes of interest and it has a range of applications such as measuring how fast a certain protein can be degraded in a cell. The ClpP sequence which was pulled from the
E. coli genome contained illegal restriction enzyme sites. In order to remove these illegal sites, wobbles were introduced into the ClpP sequence so that the gene is not cut anywhere and is allowed to function properly. ClpP is a naturally present gene in the
E. coli genome.
BBa_K1911003: ClpX
ClpX is part of the protein-degradation system called ClpXP. It works in unison with another protein called ClpXP so that it can de-linearize and unfold proteins into amino acids which can then be recycled by the cells. In
E. coli cells, ClpXP focuses on degrading incorrect proteins and reducing them down into amino acids which can be recycled by the cells. ClpXP recognizes the incorrect proteins by certain SsrA-tags which are attached at the ends of incorrect proteins. ClpX in unison with ClpP can be used to degrade unwanted proteins and can be used in future projects in conjunction with certain degradation tags. There were illegal restriction sites which cut the ClpX gene into smaller sequences, rendering it obsolete. So we had to introduce wobbles to eliminate these illegal restriction sites so that ClpX may be expressed and allowed to function as a complete protein. ClpX is found naturally in the
E. coli genome.
BBa_K1911005: eGFP
eGFP = Enhanced green fluorescent protein which can be used as a reporter gene. eGFP was used in our project to show the effects of attaching the DAS and LAA degradation tag to a target gene which is then degraded by the ClpXP system. In essence, the eGFP should be less prominent in
E. coli cells which contain DAS tags and LAA tags compared to the
E. coli cells that have constructs which do not have any degradation tags attached to the eGFP. eGFP can be used as a reporter gene and helps the observer easily see the coloration of
E. coli cells which indicate a successful transformation. The eGFP could not contain any illegal restriction enzyme sites as it would cut the sequence up, not allowing for the expression of the eGFP protein. GFP is originally found in jellyfish
Aequorea victoria but eGFP has been modified to be more sensitive. It helps the observers to easily determine the green coloration of the GFP protein.
