Revision as of 00:21, 21 June 2014 by Bklebe2016 (Talk | contribs)
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


Our original construction of our expression plasmid did not go according to plan. We received the parts and ligated them, but were confounded when our transformations of this plasmid into competent cells did not work. Thanks to the Silver Lab at Harvard Medical School, we were able to use SeqBuilder software to examine our sequences and the restriction sites to make sure that they were intact. To our dismay, we learned that a deleted base, guanine, in our sequence led to a frame shift. Since this problem was easily pinpointed, we were able to construct a new expression plasmid to be synthesized by IDT. Experiments on this new plasmid will be taking place in the coming week.

Because of this delay in our lab experimentation, the actual findings of our work are not on this Wiki, but will be available for presentation at Jamboree 2014. In this Wiki, our experimental methods and expected results will be summarized. The goal of our experimental work is to assay the culture media and the lysate of the cotransformed BK21 DE3 cells in order to detect the presence of RiAFP in these fractions. The analyzes will be run on the fractions before and after induction of the expression of RiAFP with IPTG.

A word about the induction of the system by IPTG

The lac operator sequence is incorporated into the T7 promoter. The gene for RiAFP is inserted just after the T7 promoter DNA sequence. The lac repressor (LacI) binds to the lac operator sequence and blocks T7 RNA polymerase from binding the promoter sequence. Before the gene for RiAFP is transcribed two things need to happen:
1. The lac repressor (LacI) must fall off of the operator DNA sequence in front of the RiAFP gene
2. T7 RNA polymerase must be introduced and recognize the T7 promoter in the absence of repressor.
When both of these factors are satisfied, transcription of the gene will proceed rapidly. If induction occurs during the mid-log phase of growth (OD600nm between 0.6 and 0.8), the yield of RiAFP protein will be maximized.

When lactose binds to LacI it induces a conformational change in the protein structure that renders it incapable of binding to the operator DNA sequence. IPTG is a structural mimic of lactose (it resembles the galactose sugar) that also binds to the lac repressor and induces a similar conformational change that greatly reduces its affinity for DNA. Unlike lactose, IPTG is not part of any metabolic pathways and so will not be broken down or used by the cell. This ensures that the concentration of IPTG added remains constant, making it a more useful inducer of the lac operon than lactose itself. Once the lac repressor can no longer bind the operator, native E. coli RNA polymerase begins transcribing, in high numbers, the T7 RNA polymerase gene engineered into its chromosome. Once the T7 RNA polymerase protein is expressed, it binds to the T7 promoter sequence upstream of the gene for RiAFP on the plasmid insert and transcribes the RiAFP gene.

The initiation of transcription of the RiAFP gene culminates in the secretion or RiAFP into the cytoplasm of the BL21 DE3 cells, thence into the periplasm of the cell, and finally into the space outside the cell membrane.

We use standard methods to obtain cell-free supernatant from the culture media as well as proteins from the cell lysate. Proteins from these two fractions are separated by electrophoresis on an NuPAGE Bis-Tris gel (4%–12%). The gel is stained with InVision™ His-tag In-gel Stain. This is a fluorescent stain for detection of histidine-tagged recombinant fusion proteins in protein polyacrylamide gels and is capable of detecting ~0.5 picomole of a 6X His-tagged fusion protein (e.g. 1 picomole of a 30 kDa protein is 30 ng). This technique eliminates membrane transfer and Western blotting steps.

One of the lanes of the electrophoresis gel contains BenchMark™ His-tagged Protein Standard, used as a positive control and for molecular weight sizing in his-tagged fusion protein detection. This molecular weight standard produces 10 sharp and clear bands in the range of 10-160 kDa for molecular weight estimation of his-tagged proteins (see figure of benchmark standard). The molecular weight standards are used to measure the relative sizes of the unknown proteins. RiAFP has a molecular weight (MW) of 12.8 kDa. In the lanes with samples of protein from the supernatant and cell lysate before induction with IPTG, there will be no detectable band corresponding to a protein of a MW of 12.8 kDa. However, the lanes with samples of protein from the supernatant and cell lysate after induction there should be detectable tagged protein corresponding to the expected MW of RiAFP