In 2010, Georgia Institute of Technology’s iGem team submitted the part K410000, a periplasmic heat generator made with HybB and OmpA and AOX. The following year several teams used HybB in their projects with mixed results. Our project is the continuation these projects and the characterization of HybB. To do this we will put RFP under HybB promotion. With HybB we are hoping to get reliable results from cold shock treatment.
The purpose of our project is to Biobrick HybB, which is a cold shock promoter. A Biobrick is a sequence of DNA that has standard prefixes and suffixes. The HybB promoter showed up in the Parts Registry and iGEM literature in 2005. The use of HybB has shown mixed results. Our goal is to have a reliable cold shock promoter in order to use temperature to regulate protein expression. We decided to use HybB as a promoter because, though it has only yielded mixed results, it showed promise.
HybB was synthesized for the first time by the 2005 UCSF iGEM team. The 2006 MIT iGEM team standardized HybB and submitted it to the parts registry. HybB has the part number J45503 and is not available from the Parts Registry. Georgia Tech’s 2010 iGEM team engineered a chassis to include HybB, OmpA, and AOX. The 2011 Leuven iGEM team reported, “We see that promoter activity is induced when cells are transferred to 25°C and even when they are put in an ice bath (4°C). Unfortunately, however, cells that are kept at 37°C also display an increase in promoter activity, indicating leakiness in the system.” The 2011 Groningen team reported that the HybB Promoter did not work. Through sequencing, they proved that HybB was found in the cells, and they ran the reactions at the same temperatures.However, they could not get the same results as the Leuven team. Due to these more recent findings, we are not sure whether HybB works or not.
We obtained HybB from Georgia Tech's 2010 iGEM Team, but upon sequencing and experimentation, we learned that the stock was not reliable. We then designed primers to isolate the proposed HybB promoter sequence from the ''E. coli''DH10 genome.
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