Verified successful E. coli biobrick transformants and prepared them for Sanger sequencing. New colonies were inoculated from the unsuccessful transformations. Inoculated T. cruzi and pSB1A10 with RBSs transformations.
Ran PCR with newly extracted D. rad DNA. Hopeful for success.
Sent successful E. coli biobrick samples for Sanger sequencing.
Mini-prepped second inoculations from unsuccessful transformations. Attempted new DNA extraction technique on D. rad to obtain enough DNA to run successful PCRs. Nanodrop results to follow.
Ran PCR with new D. rad DNA. Continues to be unsuccessful. We will attempt a new method of extracting D. rad DNA. Received Sanger sequencing results back for our transformed E. coli biobrick samples. Multiple successful gene insertions! Transformed the unsuccessful one again in hopes of obtaining a colony with our inserted radioresistant gene. Looking to verify that RBS's successfully ligated into pSB1A10 before we add in our successful biobricks. Digested plasmids with EcoR1 and Pst1. Will run a 2% gel and low voltage to attempt to see the 15bp RBS's with their overhangs on the gel. Identification of RBS on the 2% gel unsuccessful.
Preparing glycerol stocks of all strains that were successfully transformed with verified biobricks.
Growing up large quantities of plasmid containing RBS so we can insert a promoter upstream and our gene of interest downstream.
Also trying to use primers that amplify only the biobrick region of the plasmid, to try to avoid the need to grow up large colonies for every part - if we can amplify just the biobrick region we will be able to ligate the pieces we need directly into the plasmid (containing RBS and and promoter).
We will also be biobricking a promoter + RBS part; a brick we think will be of major use for future iGEM teams. Hard to believe the parts catalog doesn't already offer one. Anyway, having this part will enable teams to test genes as soon as they the can successfully amplify them and add the biobrick prefix and suffix. Using the promoter + RBS plasmid as the screening plasmid will mean that transformants will be expressing the gene of interest right away.