Dina:

• Email Dr. Vincent with updates
• Re-read protocols, and write them by hand into lab notebook for next day

iGEM team meeting

Interlab:

• Obtained DH5α cells (Genetics Lab 2013) from Maya
• Start preparing competent DH5α cells:

DspB Lab:

• Meeting with Dr. Vincent: changed protocol for the preparation of competent cells as Cheryl mentioned in her email, need to use ultra competent cells (from Dr. Ihab's lab) → will start next week

Biofilm Lab:

• Meeting with Bernadette and Maya to discuss materials and applinces being used in the lab for Biofilm Production.
• Meeting with Professor Vincent to discuss how to proceed with porject:
• 96 well plates.
• Add samples of SRB directly onto the wells (no inoculation); see if media is required for biofilm formation.
• Possible simulation using appratus in lab (Bioflux 2000; https://bioflux.fluxionbio.com/microbiology )

To do:

• Write up protocol for biofilm production on plastic pipe.
• SRB media recipe (just in case wemay need to use it)
• Appratus set up

Interlab:

• Finish preparing competent DH5α cells
• Prepare CaCl2 w/ Glycerol solution
• Filter sterilize all solutions

Group meeting:

• Test for DspB secretion outside after successful transformation

Skype meeting with Chyrl

Interlab:

• Competent cells test: there was no growth on any of the plates except for tiny colonies on the 100 pb/µL plate. Another method to prepare competent DH5α cells should be utilized.

Meeting to discuss Wiki with Yasmin

Biofilm lab:

• Plated flouresent DH5α were incoulated on LB Ampicilin/Arabinose broth on 50 ml kleftt flask; left over 2 days in shaker at 37°C.

DspB Lab:

• Obtain cells from Dr. Ihab lab, alongside the protocol
• Cells from competent cell test grew after ≈ 5 days

Biofilm Lab:

• Dilutions and original culture in broth were checked under UV and no fluorescence was found. Plated GFP-DH5α cells were found to be fluorescent. Cultures in broth were scrapped and new LB Ampicilin/Arabinose broth was planned to be made.

DspB Lab:

• Transform DspB (BBa_K1659200 | Kit plate 7 | well 15C) into ultra competent cells

Biofilm Lab:

• Inoculate GFP-DH5α cells in 50 ml LB Amp/Arab broth in 150 ml Kelft flask. Left in shaker at 37 overnight to culture.

Biofilm lab:

• Using spechtophotometer, OD of inoculated cultre was found to be ~ 375. 3 dilutions were made at 7.5X, 13X and 26X. Dilutions and culture were left to incubate overnight in the air shaker at 37°C.

DspB Lab:

• Overnight culture of DspB plate (from fridge) into LB Chl media, picked two colonies and left overnight shaking at 37˚C.

DspB Lab:

• Plated the remaining ultra competent cells ≈ 20 µLon LB plate to make more ultra-competent cells
• Plated 50 µL of Saad's prepared DH5 Alpha on LB plate to make more competent cells
• Transformation of DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I ) into LB-Chl plate
• Need to do this for gene expression: DspB-DspA contained in the pSB1C3 backbone, was extracted via Miniprep. The NcoI restriction site was introduced upstream of each of our gene sequences (except BBa_K1659501 and BBa_K1659601) via PCR to facilitate their insertion into the arabinose-inducible pBAD/HisB commercial expression vector, and the insert-containing expression vectors were subsequently cloned separately into the standard laboratory E. coli K-12 strain MG1655 as well as a chemotaxis knockout strain E. coli RP437 ∆FliC.

DspB Lab:

• Growth on the DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I) plate, only 4 colonies, leave over weekend
• Store other plates (cells) in the fridge

DspB Lab:

• Mini prep to extract DspB-DspA (BBa_K1659211 | Kit plate 7 | well 15I)
• Agarosegel run of DspB-DspA unsuccessful

iGEM Team meeting

Dina:

• Meeting with Oxy Qatar for human resources

Interlab Study submission

Dina and Albandari:

Dina and Kawthar:

• Double digest of pRSET plasmid with EcoRI and BamHI successful

Aisha:

• Digestion of 3 samples of DspB DspA plasmid with PstI to check size

Saad:

• Extraction of pRSET plasmid from gel: concentration = 19.8 µg/mL

Dina and Alreem:

• PCR of DspB DspA

PCR cleanup

Digestion

Cleanup

Ligation

Transformation

DspBDspA in pRSET:

• Mini prep
• Transform in BL21

ProU:

• Inoculate into LB-Chl