Difference between revisions of "Part:BBa K3737001"

Revision as of 00:48, 22 October 2021

Estrogen receptor driven by the T7 Promoter

Added IPTG to activate the RNAP to allow for the expression of the T7 promoter.

Characterizing Expression - Alma 2021

After creating this BioBrick and confirming its sequence, we wanted to find out if bacteria could grow and express it. Attempts to create other estrogen receptors, such as that from Humans, has proven to be toxic to bacteria (e.g. see part BBa_K123003)

Since this Biobrick requires the T7 polymerase for expression, we transformed it into Nico21-DE3 strain from New England Biolabs.

This strain was able to grow, and bacteria continued to grow (the OD increased) after addition of 1mM IPTG to the culture - this indicates that the gene is not toxic.

qPCR results show that the RNA is expressed - crude nucleic acid lysates were subjected to treatment with Reverse transcriptase, or with a control. Following this treatment, they were used in a qPCR with primers specific for this gene.

In a preliminary experiment, the Ct value for the treatment with RT was lower than the control - a difference of 8.13 versus 9.25; this indicates that including the RNA in the cell leads to a 2.17-fold higher amount of nucleic acid. Therefore, we conclude that this gene is being transcribed. Subsequent experiments showed that this expression is dependent on induction with IPTG.

Finally, we performed SDS PAGE on whole cell lysates for cultures with or without IPTG. Preliminary results indicate the presence of a band in a range that is expected of our gene. Together, we conclude that this part works as intended, and produces at least a bit of the estrogen receptor while allowing the bacteria to live.

Sequence and Features