Team:Glasgow/Weekly Report/Weeks 5and6

Week 5

Wet Lab

• Testing and analysis of constructs
  A lot of work was done this week to check that our constructs are correct. Sequencing of PSB1C3/GvpA/GvpC revealed a 4 basepair deletion in the GvpA RBS. We therefore decided to go back to our original glycerols of pSB1C3/GvpA and pSB1C3/GvpC.
• A restriction digest using EcoR1+ Pst1 was carried out to ensure they contained the correct insert. Another restriction digest was carried out on pZJ53B/MCS to determine whether or not the MCS was inserted correctly.
• The isolated DNA was sent for sequencing as well as J23100/GvpA/GvpC and J23100/MotA. Sequencing of MotA/pSB1C3 showed no mutation.
• A restriction digest revealed that the Top10 transformaiton of J23100/GvpA/GvpC contain a mix of plasmids. This was confirmed by testing uncut DNA, single digest and a double digest.
• Sample #1 which contains the desired DNA was then transformed into Top10, DH5α and DS941. Some of the resultant transformations were miniprepped and digested (EcoR1 + Pst1) – this showed that the mixture of DNA had separated in the transformants. Unfortunately, the single DS941 transformed did not contain the desired DNA. Restriction digests of the transformants from the Top10 and DH5α strains revealed which transformants contains the desired plasmid.
• FliC
  PCR was carried out to isolate the FliC gene; this was to be used in a topisomerase reaction to remove a restriction site within the gene. Unfortunately, the topisomerase reaction was unsuccessful – this may be due to a faulty kit.
• MotA Knockouts
  The swimming ability of the MotA KO strain was compared to the WT strain using “sloppy” agar, revealing that the Kos were unable to swim). The KO was also transformed with the MotA plasmids (J23100/MotA and pSB1C3/MotA) to test the ability of the plasmid to rescue swimming.
• GFP
  In order to prove the presence of GFP is due to the promotor changing direction in response to integrase, colonies were short streaked. Streaks were then selected to be miniprepped, and digested and compared on a gel.
• Four colonies which fluoresced brightly, four which did not fluoresce and two which had intermediate fluorescence were selected. Restriction digest with HindIII and Pst1 revealed that the bright colonies contained a switched promoter whereas the non-fluorescing colonies do not. The intermediate colonies contained either both switched and non-switched plasmids or very little switched DNA.
• After establishing that the switch is successful, oligos were designed for the reverse components of the switch: reverse RFP, reverse MotA and the different reverse RBS. Oigos were also designed to allow Phi C31 integrase and Gp3 to be made into biobricks.

Dry Lab

• All the components required for the gas vesicle measurement apparatus was assembled.
• A lot of work was also done on the wiki design: including the front page.

Admin and Outreach

Figure 1: Chocolate Brownies (baked by Lydia)

Week 6

Wet Lab

Dry Lab

Admin and Outreach

Figure 2: Flapjack Cookies (baked by Martin)