Team:Groningen/Parts/Used Parts

iGEM Groningen 2009

The ligation of part behind the RBS was successful, confirmed by gel (correct vector size after digestion with EcoRI and PstI) and sequencing with VF2 primer. We used this part in combination with several biobricks for building our constructs e.g. .

iGEM Groningen 2009

The plasmid containing part was successfully transformed into E. coli TOP10 cells (confirmed by single and double digestion). The original plan was to use the terminator behind one of our own designed parts , and in front of pArsR-RFP. The MBP-ArsR fusion protein was thought to have a regulating effect on the arsenic promotor pArsR in the same way as ArsR regulates the promotor. The terminator separated the two parts on the plasmid. The construct was not used due to time constraints, and not sequenced.

iGEM Groningen 2009

Part was one of our main focus points during our project. The part was originally submitted by Melbourne in 2007 with a very limited amount of information (short description and sequence mutations). On their site it was mentioned the cluster was difficult to use and ligation into a plasmid was hard, in contrast we found the cluster to be easily cut and ligated into different plasmids and behind several promotors. We used this part in combination with several biobricks for building our constructs e.g. and . Pictures of our cells with induced gas vesicle formation confirmed the production of vesicles in the expected shape and size. In addition, we characterized the part to improve the use in the future and added a lot of information on the registry.

iGEM Groningen 2009

P1010 is used when putting BioBrick parts into BioBrick plasmids. The part to be inserted and the plasmid are cut with BioBrick enzymes and mixed. The mixture will include both the original uncut or self ligated plasmid and the desired structure. However, because of CcdB, all of the cells containing the original plasmid die and the surviving colonies are the desired result. The ccdB cell death gene worked as expected killing our E. coli TOP10 cells, and keeping our E. coli DB3 cells alive. After noticing the inconsistent sequencing result for the pSB2K3 plasmid with ccdB cell death gene, we decided to choose a different pSB2K3 plasmid with random part to continue with our assemblies. This to minimize the chance of unwanted surprises in the end.

iGEM Groningen 2009

pSB1AC3 is a high copy number plasmid carrying ampicillin and chloramphenicol resistance. Together with pSB1A2 the vector was used for most assemblies of our team, because the high copy number made it easy to work with and easy isolation. The transformations with pSB1AC3 (containing different biobricks of own design) into E. coli TOP10 cells, growth on both antibiotics, and gel analysis (undigested and digested with the EcoRI and PstI) worked as expected. The high (copy) number of plasmids per cell make it an easy to work with plasmid, ideal for cloning and assembly work.