Difference between revisions of "Part:BBa K3174002"
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<figcaption><b>Fig.1:</b> 5 different BFP plasmids in TOP10 cells grown in a 5mL O/N culture viewed under a UV light: (left to right) BBa_K3174002, BBa_K3174003, BBa_K3174004, BBa_K3174006, and BBa_K3174007. The same streaking method was used for all 5 of these plates. BBa_K3174002 and BBa_K3174003 have very small colonies. BBa_K3174004, and many for BBa_K3174006 and BBa_K3174007. The disparity in growth rates originates from the variation in metabolic burden imposed on each strain due to their relative promoter and RBS strengths.</figcaption>
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<figcaption><b>Fig.1:</b> 5 different BFP plasmids in TOP10 cells streaked onto LB + CAM plates from glycerol stocks, and viewed under a UV light: (left to right) BBa_K3174002, BBa_K3174003, BBa_K3174004, BBa_K3174006, and BBa_K3174007. BBa_K3174002 and BBa_K3174003 have very small fluorescent colonies, BBa_K3174004 has bigger colonies, and BBa_K3174006 and BBa_K3174007 have the largest colonies. The disparity in these growth rates originates from the variation in metabolic burden imposed on each strain due to their relative promoter and RBS strengths.</figcaption>
 
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<p>The differential growth rate observed between these 5 parts results from different promoter and RBS combinations that each impose a proportional level of metabolic burden on the host cell. BBa_K3174002 and BBa_K3174003 both have strong promoters, therefore, they grow very few single colonies when streaked on a plate and express the lowest level of BFP because burdened cells are quickly outcompeted by the more fit mutated cells which contain broken genetic circuits. They should be used with caution because they break almost immediately due to this stress. BBa_K3174004, containing a strong promoter and weak RBS, grows more single colonies than BBa_K3174002 and BBa_K3174003 and expresses BFP at the highest level of these 5 parts because it is the most stable. BBa_K3174006 and BBa_K3174007 grow the most single colonies and express BFP at a higher average level than BBa_K3174002 and BBa_K3174003 but at a lower level than BBa_K3174004.</p>
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<p> 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. 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 <b>lowest level of BFP expression</b>. 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.</p>
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<p>BBa_K31740064 seems to be the most stable of these 5 parts and is recommended for experiments that rely on evolutionary stability.</p>
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<p> 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.</p>
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<p> 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.</p>
  
 
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<p> The major takeaways from this measurement data are:
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<li> 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.</li>
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<li> 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.</li>
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<li> 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.</li>
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Revision as of 19:44, 16 October 2019


Blue Fluorescent Protein with Strong Promoter and Strong RBS

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

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

BBa_B0034 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.

Fig.1: 5 different BFP plasmids in TOP10 cells streaked onto LB + CAM plates from glycerol stocks, and viewed under a UV light: (left to right) BBa_K3174002, BBa_K3174003, BBa_K3174004, BBa_K3174006, and BBa_K3174007. BBa_K3174002 and BBa_K3174003 have very small fluorescent colonies, BBa_K3174004 has bigger colonies, and BBa_K3174006 and BBa_K3174007 have the largest colonies. The disparity in these growth rates originates from the variation in metabolic burden imposed on each strain due to their relative promoter and RBS strengths.

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. 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.

Fig.2: BFP expression for 3 isolates of each of the 5 parts with. The standard deviation for each bar BBa_K3174002 and BBa_K3174003 have the lowest BFP expression levels because they have mutated as a result of high metabolic stress. BBa_K3174004 has the highest BFP expression level because it is stable and contains a strong promoter. BBa_K3174006 and BBa_K3174007 have intermediate BFP expression levels because they are stable but contain weaker promoter/RBS combinations.

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 (https://2019.igem.org/Team:Austin_UTexas) 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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]