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Team:BostonU/NotebookQS
From 2013.igem.org
Quorum Sensing Notebook
June
We planned out that we will transform E. coli with the civI/civR system found in Chromobacterium violaceum. We will design MoClo devices that include these systems. In the meantime, we are beginning to design primers that will make the civI/civR system into a MoClo system by adding the the fusion site in addition to the BBS and SpeI sites. Then, we will develop a PCR strategy.
Our advisor, Traci, emailed different labs and PIs that work with Chromobacterium violaceum. Dr. Jim Thoden from the Holden Lab at the University of Wisconsin sent us genomic DNA.
We designed forward and reverse primers for CviR/CviI systems and ordered those primers. We are currently waiting on acquiring the primers to begin PCR.
July
Today, we did Phusion PCR using the genomic DNA of Chomobacterium violaceum as the template for the CviI and CviR systems. We ran the PCR product on a gel and the CviI did not appear on the gel. The CviR system appeared and we gel extracted the parts.
The gel concentrations are as follows:
We redid the PCR for CviI. We ran the PCR product on a gel and we did the gel extraction. we quantified the concentration on the NanoDrop.
We ran a Level 0 MoClo reaction with the higher concentration of CviI and CviR.
We did a transformation using Bioline Gold E. coli and plated the samples on IPTG + Xgal.
We pulled the plates and picked three colonies and plated them on a stab plate and set up liquid cultures for incubation.
We set up CviI_CD and CviR_CD for sequencing in triplicate.
We analyzed the sequences for CviI and CviR and all of the samples we sent in matched the expected sequence.
We made glycerols and archived the highest concentration of each CviI and CviR.
Traci ordered the promoter pVioA_AB from IDT. It should come in from 4-10 business days. We are to treat it as PCR product and do L0 MoClo reaction. We can build the following four Level 1 devices and combine them together for a Level 2 device.
We are still waiting for the pVioA promoter. In the meantime, we are making I13453_GB (pBAD)and sending four samples out for sequencing. Here is an image of its transformation.
One of the I13453_GB samples were a match so we can make glycerol stocks and miniprep plasmid and archive the parts in our registry.
We ran Level 1 MoClo reactions for:
- E-J23100_EB-BCD2_BC-CviR_CD-B0015_DF-F
- F-I13453_FB-BCD2_BC-CviI_CD-B0015_DG-G
- G-I13453_GB-BCD2_BC-XFP_CD-B0015_DH-H
Today we transformed yesterday’s reactions using Bioline competent cells and plated the transformations on IPTG and Xgal for blue white screening.
August
We picked white colonies for overnight cultures.
The cultures were miniprepped and sent for sequencing but they were unfortunately wrong.
pVioA finally arrived so we can create four transcriptional units using MoClo. First we had to make the Level 0 part for pVioA promoter in the AB an EB format.
With the sequence of pVioA_AB and pVioA_EB verified, we set up the level 1 reaction for:
- pVioA_AB+BCD2_BC+E1010_CD+B0015-DE+DVL1_AE
- J23100_EB+BCD2_BC+CviR_CD+B0015_DF+DVL1_EF
- I13453_FB+BCD2_BC+CviI_CD+B0015_DG+DVL1_FG
- I13453_GB+BCD2_BC+E0040_CD+B0015_DH+DVL1_GH
After sequencing, we seem to have had a mix up of promoter and CDS. CviR is paired with I13453 instead of J23100, while CviI is paired with J23100 instead of I13453. Other lab mates are redoing the reactions today to ensure no other errors occur.
The Level 1 reactions were redone, checked on a gel, and sent for sequencing because last week's reactions were unsuccessful.
We did the sequence analysis for the Level 1 parts and set up glycerol stocks.
We realized that we will need a fifth Level 1 reaction to incorporate arabinose as a small molecule. We will do this using the araC CDS. We also decided that we are going to change the J23100 promoter to J23104. The reactions are now as follows:
- QS1 = pVioA_AB+BCD2_BC+E1010_CD+B0015_DE+DVL1_AE
- QS2 = J23104_EB+BCD2_BC+CviR_CD+B0015_DF+DVL1_EF
- QS3 = I13453_FB+BCD2_BC+CviI_CD+B0015_DG+DVL1_FG
- QS4 = I13453_GB+BCD2_BC+E0040_CD+B0015_DH+DVL1_GH
- QS5 = J23104_HB+BCD2_BC+C0080_CD+B0015_DJ+DVL1_HJ
- QS6 = QS1+QS2+QS3+QS4+QS5
Also did PCR and gel extraction for C0061_CD, C0062_CD and R0062_CD. They are parts necessary for the LuxI/R quorum sensing that is theoretically orthogonal to the C. violaceum system being put together.
We analyzed the sequences for C0061_CD and C0062_CD. C0062_CD was confirmed but the C0061_CD sequence included only one of the PCR product inserts.
