This marked the last week of this research experience! We did not stop experimentation, however!

Key highlights of this week:

Monday

Today, we began the process of IPTG induction. In this process, a gene coding for the desired protein is inserted into E. coli and placed under the control of the lac operon, a natural regulatory system that controls the expression of certain genes in response to the presence of lactose. IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a synthetic analog of lactose that cannot be metabolized by E. coli. When IPTG is added to the culture, it binds to the repressor protein that normally keeps the lac operon turned off, causing the repressor to release from the DNA. This release allows RNA polymerase to bind to the promoter region of the operon and initiate transcription of the gene, leading to the production of the target protein. IPTG induction is favored for its ability to precisely control the timing of protein expression, ensuring that the bacteria can grow to a sufficient density before protein production begins, which often results in higher yields and more efficient use of resources.

We began by making LB broth and autoclaving it. Once the LB was made, we let it cool. Then, we made overnight cultures in 50 mL tubes using previously made LB, stored at the bench. These cultures were done as per usual: carbenicillin and chlorophyllin were added to LB as selective antibiotics. Then, one colony was picked off of a previously transformed plate of E. coli, transformed with Levin MCP and construct 9337. These two overnight cultures were incubated in a 37 degree shaking incubator overnight.

Tuesday

Two flasks with LB were inoculated with the liquid cultures from the previous day and stored on the shaking incubator in the environmental warm room until the optical density reached 0.5-0.6. This was determined by regular checking of the OD of these two flasks. Once it reached this OD value, we carried out the process of IPTG induction. We first made 3 mL of 1 M IPTG. Then, we pipetted in 1 mL of IPTG into both of the flasks and stored them overnight in a shaking incubator at 16 degrees and 220 RPM. This marked the end of Tuesday’s work. As a side note, a lot of this week went into practicing presentations.

Wednesday

We prepared DNA for sequencing today. It involved a lot of new procedures and also involved PCR. After the plasmid minipreparation, a standard lab procedure, was complete, we tested the cell count using a nanodrop, and when we determined it was sufficient, we sent it across the street for sequencing at the Med Center.

Moreover, we pelleted, lysed, and spun the cells from yesterday. We then took samples of the supernatant and pellet. We also washed our nickel column with 8 M urea and stored the supernatant and beads in a 50 mL conical tube for elution tomorrow.

Thursday

Today, we eluted the protein using 2 concentrations of imidazole: 50 mM and 300 mM. The 50 mM served as a wash, while the 250 mM served as an elution. Using standard Ni-NTA protocol, we washed and eluted the protein, took samples of everything (including the supernatant and pellet from yesterday), and ran an SDS-PAGE gel. The results were overnight, so we will find out tomorrow whether or not it succeeded in purifying the protein an adequate amount.

I am very excited for the presentation tomorrow! Good luck everyone!