Team:UCLouvain/Composite Part

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 Composite parts

GFP coding device

This biobrick was used as a reporter for our donor cells during the conjugation. We chose to work with Bba_E0840 because it contained a weak RBS, and so it wouldn’t weaken the transcription of essential genes and/or of our low copy conjugation plasmid.

Figure 1: GFP coding device

This device is composed of two subparts:

• LacI regulated promoter Bba_R0010
This promoter comes from the Lac operon. It’s a strong promoter in absence of the expressed LacI gene. If this gene is expressed, the LacI protein can be inhibited by lactose or his non metabolizable homolog, IPTG.
• GFP generator Bba_E0840 Bba_R0010
This part consists in a weak RBS (Bba_B0030) ), a GFP gene (Bba_E0040) and two terminators (Bba_B0010 , Bba_B0012 ).

Reporter Plasmids

Figure 2: Bactwars : Leila Skydonor (in green) and Dark Receiver (in red) before the fight

RFP Reporter – Receiver Cells

As a proof of concept, our idea was to target a gene encoding for a red fluorescent protein carried on a low copy plasmid in the recipient cells. Those plasmids were available in the iGEM 2018 DNA distribution kit as a composite part.

To perform our experiments, we studied three parts with different resistance genes :

• pSB4C5-J04450
• pSB4K5-J04450
• pSB4A5-J04450

Figure 3: RFP reporter plasmid design used in receiver strains

We further characterized the pSB4K5_J04450 system by studying its lifetime in DH5α. This allowed us to study whether the fluorescence loss was due to the natural loss of the plasmid or the cut made by our CRISPR Cas9 system. See the results

GFP Reporter – Donor Cells

In order to identify the donor cells, we designed a reporter plasmid with a low copy number which we transformed into our donor cells. This DNA sequence was designed using different iGEM part with the promoter Bba_R0010, the GFP coding device Bba_E0840 and the low copy plasmid backbone pSB4C5 .

Figure 4: GFP reporter plasmid design used in donor strains

See the experiments

Reference:
Engineering BioBrick vectors from BioBrick parts
Journal of Biological Engineering, 2008 Apr 14;2:5
Reshma Shetty, Drew Endy, Tom Knight