Difference between revisions of "Part:BBa K3174004"

Revision as of 19:50, 21 October 2019

Blue Fluorescent Protein with Strong Promoter and Weak RBS

This part has the blue fluorescent protein (mTagBFP) gene inserted downstream of a strong promoter and weak ribosome binding site.

BBa_J23104 is part of the Anderson Series of constitutive promoters designed by iGEM2006_Berkeley.

BBa_B0031 is an RBS based on the Elowitz repressilator and was designed in 2003. Its relative strength was characterized by IIT Madras in 2016 and by Team Warsaw in 2010.

BBa_K592100 is a blue fluorescent protein designed by iGEM11_Uppsala-Sweden in 2011. It has a maximum emission of 456 nm.

In order to visualize the growth rate of these parts, the 5 glycerol stocks were streaked out onto LB + CAM plates and grown in a 37 degree incubator for a period of 24 hours. Fig.1 below shows the results.

Once we had observed growth rates of each of these 5 parts, we wanted to see if the BFP expression levels matched our expectations based on what we know about the promoter strength of each part. We did this by selecting 3 colonies from each of these 5 plates and growing them in 5mL overnight LB + CAM cultures. We then placed 4 replicates of each isolate into a 96 -well plate (using 200uL of culture for each well) and took a single BFP measurement for each well. The resulting data is depicted in the Fig.2 bar plot below.

We expected BBa_K3174002 and BBa_K3174003 to have the highest BFP expression levels because these parts have the strongest promoter/RBS combinations. Surprisingly, we found that they had the lowest level of BFP expression. We hypothesized that the cells with the intact genetic devices must have been under a high level of metabolic stress, resulting in loss-of-function mutations that contained a 'broken' genetic device (making these cells express lower levels of BFP). The new mutated cells, with their fitness advantage over the highly burdened intact cells, must have then quickly overtaken the population.

We expected BBa_K3174004 to have a BFP expression level that was lower than BBa_K3174002 and BBa_K3174003 but higher than BBa_K3174006 and BBa_K3174007. However, it instead had the highest level of BFP expression out of all 5 parts. This is probably due to the fact that it had a strong enough promoter to cause BFP to be expressed strongly but a weak RBS that prevented considerable stress to the cell. It is important to note that although BBa_K3174004 expressed BFP at the highest level, it lacked consistency between each of the four replicates for each isolate.

BBa_K3174006 and BBa_K3174007 expressed BFP as expected. They had the second and third highest BFP expression levels respectively. Both these parts have relatively weaker promoters that are unlikely to cause burden to the cell. They seemed to have the highest consistency between replicates.

These 5 constructs had their sequences confirmed via Sanger Sequencing.

The major takeaways from this measurement data are:

• BBa_K3174002 and BBa_K3174003 should be used with caution because they break almost immediately due to a high level of metabolic stress to the cell that causes loss of function mutations, leading to a population of cells that no longer express BFP at a high level.
• BBa_K3174004 expresses BFP at the highest level but probably does not have very high stability based on the inconsistency in BFP expression measurements between replicates of each of the three isolates depicted in the bar graph above.
• BBa_K3174006 and BBa_K3174007 express BFP at low levels but appear to be the most stable based on the consistency in BFP expression measurements between replicates of each of the three isolates depicted in the bar graph above. These parts could be useful for experiments that do not require high levels of BFP expression but do rely on evolutionary stability.

Usage and Biology

UT Austin iGEM 2019 Team transformed this part into the E.coli DH10B burden monitor and used it as a standard with which cellular burden could be quantified (by means of a GFP expression vs growth rate model) for other biobricks that had been similarly transformed into the burden strain. For more information on characterization of these parts through the burden monitor, visit our team’s wiki page: [1]

Sequence and Features