Team:NTNU Trondheim/Parts

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Parts overview

The team has submitted two parts to the iGEM parts registry: dCas9 regulated by a TetR inducible promoter and RRvT with lacI promoter:

Table 1:Parts submitted to the iGEM Parts Registry

Medal	Part name	Type	Description	Length (bp)
Silver	BBa_K2754000	Composite	dCas9 regulated by a TetR inducible promoter	4978
Silver	BBa_K2754001	Composite	RRvT with lacI promoter	1908

dCas9 regulated by a TetR inducible promoter

The new biobrick that we submitted to the iGEM Registry was a dCas9 sequence with a tetracycline inducible promoter (= TetR/TetA). The part also includes a TetR sequence upstream of the dCas9 and the promoter sequence, which is required for controlling the inducibility of the promoter. You can read more about the function of this part in the Design page.

The original sequence was ordered from Addgene, but we noticed that it did not meet the iGEM requirements for new biological parts due to having three illegal restriction cut sites. We therefore needed to remove these cut sites by switching some nucleotides while ensuring that the amino acid sequence remained the same. In addition, we also needed to include a specific prefix sequence in front of the biobrick and a suffix sequence at the end. This was necessary to be able to ligate the biobrick into a the pSB1C3 backbone that iGEM requests that we submit the biobrick in. The elimination of the illegal cut sites and the insert of prefix and suffix was done by Gibson Assembly and Site-Directed Mutagenesis. After this, we had to cut the biobrick and the plasmid backbone with specific restriction enzymes which have cut sites in the prefix and suffix.

Figure 1: Overview of the dCas9 biobrick

RRvT with lacI promoter

One of the gold medal criteria for the iGEM Competition is improving a biobrick that already exists in the iGEM Registry. The new and improved part also needs to be characterized to document that it is indeed better than the original part, as well as submitted to the iGEM Registry. To fulfill this criterion, we decided to improve a biobrick encoding the red fluorescent protein mRFP1 (BBa_J04450) designed by Tamar Odle. The original biobrick is a composite part with a lac promoter, a ribosome binding site, a coding sequence for the mRFP1 protein and a terminator. The improvement was done by replacing the mRFP1 sequence with a sequence for another red fluorescent protein, called RRvT.

Figure 2: The improved biobrick was designed by replacing the mRFP sequence with the sequence for the RRvT.

The RRvT is supposed to be brighter than the original mRFP1, and it also has a low acid sensitivity which theoretically should make it an improvement of the mRFP1 biobrick. We have ordered and received the RRvT biobrick from IDT. After failing to incorporate the RRvT biobrick into the iGEM plasmid backbone through traditional restricting cutting and ligating, we ordered primers for inserting the biobrick into the backbone by Gibson Assembly. The RRvT sequence and the backbone have been PCR amplified with the Gibson primers and assembled by Gibson Assembly. It was then transformed into E.coli DH5α cells. A plate reader was used to create a growth curve showing the development of absorbance and fluorescence in the cells over time, and a flow cytometer to measure light intensity per cell. The measurement of our new biobrick was done in paralell with the original mRFP1 biobrick to ensure that both parts have a similar experimental environment. This made the comparison between the two more accurate. Unfortunately, the improvement did not work as intended. We could not characterise the improvement. However we obtained fluorescence data from the biobrick.

Figure 3: Overview of the RRvT biobrick