Lab Skills – Sean H. Geiger

The purpose of this page is to showcase the various work I’ve done in the lab. It will likely be updated and refined over time, think of it as indefinitely under contruction. If you’re interested in learning how to do something posted here—or you’d like to know the certain methods I followed—feel free to check out my Protocols section or shoot me an e-mail. Each picture will eventually contain a link to its respective protocol.

PCR of tetracycline (tet) promoters using Herculase DNA polymerase. Plasmid template (10ng/uL), run on a 1% agarose gel (recycled). — PCR of various tetracycline (tet) promoters using Herculase DNA polymerase. Amplified from a plasmid template (10 ng/uL), run on a 1% agarose gel (recycled).

A: Epifluorescence micrographs of a filament of N. punctiforme expressing a transcriptional reporter plasmid. GFP inclusions are highlighted as red arrows—1000x magnification. [B] Same filament as [A] but captured with lower exposure settings. [C] Autofuorescence of filament in [A]. [D] Wild-type (WT) filament of N. punctiforme which does not contain the transcriptional reporter plasmid. Exposure settings are equal to micrograph [A]. — **[A]** Epifluorescence micrographs of a filament of *N. punctiforme* expressing a transcriptional reporter plasmid. GFP inclusions are highlighted as red arrows—1000x magnification. **[B]** Same filament as [A] but captured with lower exposure settings. **[C]** Autofluorescence of filament in [A]. **[D]** Wild-type (WT) filament of *N. punctiforme* which does not contain the transcriptional reporter plasmid. Exposure settings are equal to micrograph [A].

[A], [B], [C], [D], [E] Various filaments of N. punctiforme expressing a translational reporter plasmid fusing a gene of interest to the gene encoding cerulean fluorescent protein (CFP). Nearly all filaments expressing this protein chimera show compromised cell-cell contacts and polar localization of the protein. — **[A], [B], [C], [D], [E]** Various filaments of *N. punctiforme* expressing a translational reporter plasmid fusing a gene of interest to the gene encoding cerulean fluorescent protein (CFP). Nearly all filaments expressing this protein chimera show compromised cell-cell contacts and polar localization of the protein. All images taken using epifluorescence microscopy, 1000x magnification.

[A], [B], [C] Filaments of N. punctiforme expressing the same translational fusion protein as in the series of images above. Filaments were stained with the neutral lipid-staining dye BODIPY. Neutral lipids appear as bright inclusions in the center of most cells. [D] WT N. punctiforme stained with BODIPY — **[A], [B], [C]** Filaments of *N. punctiforme* expressing the same translational fusion protein as in the series of images above. Filaments were stained with the neutral lipid-staining dye BODIPY. Neutral lipids appear as bright inclusions in the center of most cells. **[D]** WT *N. punctiforme* stained with BODIPY. 1000x magnification

[A], [B], [C] BODIPY-stained filaments of N. punctiforme over-expressing a protein of interest. Notice the size, aggregation, and positioning of the neutral lipids. Proteins are over-expressed by cloning the genes encoding them into a high-copy plasmid which yields around a 100-fold increase in protein expression. [D] WT N. punctiforme stained with BODIPY — **[A]** WT *N. punctiforme* stained with BODIPY. **[B], [C], [D]** BODIPY-stained filaments of *N. punctiforme* over-expressing a protein of interest. Notice the size, aggregation, and positioning of the neutral lipids. Proteins are over-expressed by cloning the genes encoding them into a high-copy plasmid which yields around 100-fold increase in protein expression. 1000x magnification.

Comparison of WT and mutant filaments of N. punctiforme. — Comparison of WT and mutant filaments of *N. punctiforme.* **[A]** Translational reporter strain of a certain gene/ protein of interest. **[B]** Mutant of the same gene/ protein targeted in translational reporter strain [A].

SDS-PAGE of a protein of interest expressed in the vector pET28a. The protein was induced for 0, 2.5, 5, and 24 hours. The protein being induced/ isolated was found to be insoluble and produced only 24 hours after induction with IPTG (red box). T = total pellet, S = soluble fraction. L = ladder. — SDS-PAGE of a protein of interest expressed in the vector pET28a. The protein was induced for 0, 2.5, 5, and 24 hours. The protein being induced/ isolated was found to be insoluble and produced only 24 hours after induction with IPTG (red box). T = total pellet. S = soluble fraction. L = ladder.

[A] Homology model of a certain protein of interest. Ribbon-only. [B] Ball and stick model of [A]. [C] Merge of [A] and side chains-only of [B]. [D] Hydrophobicity plot layered over image [C], blue represents hydrophilic residues while red represents hydrophobic residues. All images generated using UCSF Chimera software. — **[A]** Homology model of a certain protein of interest. Ribbon-only. **[B]** Ball and stick model of [A]. **[C]** Merge of [A] and side chains-only of [B]. **[D]** Hydrophobicity plot layered over image [C], blue represents hydrophilic residues while red represents hydrophobic residues. All images generated using UCSF Chimera software.

Sequencing of purified plasmids using unique primers. Sequencing provided by Laragen Sequencing & Genotyping, UCLA, Los Angeles. — Electropherogram of purified plasmid sequences using unique primers. Sequencing service provided by Laragen Sequencing & Genotyping, UCLA, Los Angeles.

Various_media — Various strains of *N. punctiforme* in Allen and Arnon media.

Thin-layer chromatography of a few different strains of Nostoc punctiforme—wild type, mutant, and overexpression. — Thin-layer chromatography of a few different strains of *N. punctiforme*—wild type, mutant, and overexpression.

Example of bacterial two-hybrid screen results. BTH101 E. coli are plated on M63 minimal media, supplemented with X-GAL, IPTG, and maltose as a carbon source.

Growth curve comparing various strains I created in the lab. One experimental strain (0288-CFP) shows a clear deviation in growth—at 17 days—compared to WT and pSUN-CERC control strains. This deviation implies that genetic modification hinders the cell’s ability to survive in stationary phase.

Immunostained filaments of N. punctiforme expressing empty FLAG vectors (Top 3) in early log phase, late log phase, and stationary phase. No fluorescence is seen.

Immunostained filaments of N. punctiforme expressing FLAG-tagged proteins (Bottom 3) in early log phase, late log phase, and stationary phase. Polar localization can be seen in stationary phase cells (red arrows).