Team:Pittsburgh/Notebook

Notebook

Virtual meetings conducted to conceptually develop our project.

Took inventory of lab supplies

Grew the streaked out plates for pMAL_beta_gal. Plasmid obtained from Dr. Xiaodong Zhu’s lab.

Made liquid culture.

Mini prep the pMAL-beta-gal

Ran a PCR for the pCZ10221 with 1% agarose gel at 120V for 45 minutes then extracted DNA from the gel.

Figure 1. 6/15/21 PCR gel for pCZ10221

Ran the PCR for pCZ10221

Figure 1. 6/15/21 PCR gel for pCZ10221

Treated the PCR product with Dpn1, Made a 1x TAE buffer, Ran the 1% gel at 120V for 30 minutes, Extracted DNA from the gel, and the DNA concentration is 52.8 ng/uL

Figure 2. 6/23/21 PCR gel for pCZ10221

Transformed pMAL_beta_gal into BL21 cells.

The plate with just Amp+ grew but the IPTG/X-gal plate did not grow. Made liquid culture.

Figure 3. 6/28/21 BL21 colonies

Made the glycerol stocks and liquid culture for plasmids from Dr. Robert Shank’s lab.

Designed pUC18_grxA, pUC18_katG, and pUC18_dps which contain OxyR binding sites.

Ordered the OxyR binding sites plasmids from genscript

Transformed pUC18_grxA into Dh5 alpha and grew on Amp+ plates.

Figure 4. 7/26/21 Dh5α colonies

Ran initial H₂O₂ induction experiment using the lysis buffer. The blue pigment was not created from X-gal.

Figure 5. 7/30/21 Lysed cells following H₂O₂ exposure

MiniPrep the pUC18_grxA plasmid and ran a restriction digest.

Figure 6. 8/3/21 Restriction Digest Gel

Transformed pUC18_grxA, pUC18_katG, and pUC18_dps into Dh5α cells, grew overnight culture

Figure 7. 8/4/21 Dh5α colonies.

MiniPrep the plasmids and ran restriction digestion.

Figure 8. 8/6/21 restriction digest gel

Ran H₂O₂ induction through incorporating the H₂O₂ into the agar plates. No blue colonies were detected.

Ran H₂O₂ plate experiment again. No blue colonies.

Figure 9. 8/13/21 Dh5α cells on Amp⁺, X-gal, and

Streaked out cells from Dr. Shank’s lab.

Designed and ordered pRSF_His_FimH and pRSF_SNAP_His_FimH from gen script.

Made BL21 competent cells.

Transformed the pRSF_His_FimH and pRSF_SNAP_His_FimH into BL21 pLysS cells.

Figure 10. 9/3/21 BL21 cells on Kan+ plates

MiniPrep pRSF_His_FimH and pRSF_SNAP_His_FimH plasmids and sent for sequencing.

Ran H₂O₂ induction using a well plate and ONPG instead of X-gal. No yellow color was detected.

Ran H₂O₂ induction using a well plate and ONPG instead of X-gal again. No yellow color was detected.

Continued to optimize H₂O₂ experiment.

IPTG on BL21 cells containing pRSF_His_FimH and pRSF_SNAP_His_FimH. Then ran an SDS-PAGE gel.

Performed nickel bead assay.