Difference between revisions of "Part:BBa K1475000"
 
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Protein containing all the essential amino acids in the right ratio, accordig to WHO (see references) and also containing all the non essential amino acids.
 
Protein containing all the essential amino acids in the right ratio, accordig to WHO (see references) and also containing all the non essential amino acids.
 
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Note this par was never submitted due to an ilegal XbaI site, characterization was done anyway because this deviation from the BioBrick standard does not affect the function of the construct.
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===Characterization===
 
===Characterization===
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The western blot was made on E. coli K12 MG1655 wild-type and E. coli expressing OneProt at different OD measures. In order to detect the protein, we probed the membrane with antibodies specific for the 3xFLAG tag contained in the OneProt sequence.
+
The protein is self-designed, so we wanted to test if the protein was expressed in ''E. coli'' K12 MG1655, by the use of Western blotting. The Western blot was blottet with ''E. coli'' K12 MG1655 wild-type and ''E. coli'' expressing OneProt at different OD600 measures.
 
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[[File:SDU2014 OneProt western.png]]
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[[File:SDU2014 OneProt western.png|400px|thumb|left|Western blot showing ''E. coli'' wild-type at OD600 at 0.3, 0.8 and 1.8 and ''E. coli'' expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.]]
 
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Western blot showing E. coli wild-type and E. coli expressing OneProt at OD at 0.3, 0.8, 1.8 and an overnight culture. The membrane was exposed for 10 minutes.
 
 
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Since bonds can be detected after probing with the specific antibodies, OneProt is expressed. However, from this western blot, we cannot see if the protein has been cut, just that it is expressed.
 
 
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In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-page. Here we also wanted to receive information on the expression of the protein at different growth stages of E. coli. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, E. coli with an empty vector (PSC1C3) was used.
 
 
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[[File:Coomassie oneprot.PNG]]
 
 
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OneProt has a molecular weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.
 
 
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To test how what effect the expression of OneProt have on E. coli we set up a growth experiment measuring OD over time on the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector.
 
 
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[[File:Growth oneprot..png]]
 
 
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Growth curve illustrating the growth of E. coli K12 MG1655 WT, odor-free E. coli YYC912, E. coli K12 expressing OneProt and with an empty vector.
 
 
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From the growth curve, it is shown that the expression of OneProt stresses the metabolism a lot compared to the E. coli K12 wild-type. In addition to this, the metabolism of YYC912 is also quite stressed compared to the K12 wild-type. Despite the stressed metabolism of the two strains, the expression of OneProt increases over time as does the growth of YYC912.  
+
The protein has a 3xFLAG tag and since bonds are showing under incubation with anti-FLAG, OneProt is expressed. However, from this Western blot, we cannot see if the protein has been cleaved, just that it is expressed.
 
<br>
 
<br>
 
<br>
 
<br>
−
Comparing the growth curve of E. coli K12 expressing OneProt, TetR(+LVA), TetR(no LVA) and odor-free E. coli YYC912 it is seen that the metabolism of E. coli expressing OneProt and TetR is stressed compared to the wild-type which means that it might be difficult to have OneProt expressed in high amounts controlled by pTet (+/-LVA). It is, however, shown that the cells are growing in despite of their stressed metabolism and 8+it is possible that the expression of OneProt can be controlled by pTet controlled by TetR although the TetR(+LVA) seems more favorable. It can also be seen that the growth of E. coli YYC912 is comprised compared to the K12 wild-type which also contributes to possible difficulties in expressing OneProt in the odor-free YYC912 strain. However, the possibility still exists.
+
 
 +
In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-PAGE. Here we also wanted to receive information on the expression of the protein at different growth stages of ''E. coli''. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, ''E. coli'' with an empty vector (PSC1C3) was used.
 
<br>
 
<br>
 +
[[File:Coomassie oneprot.PNG]]
 
<br>
 
<br>
−
[[File:Double growth curve - revideret.PNG]]
+
OneProt has a molecular weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.
 
<br>
 
<br>
−
Growth curves showing E. coli K12 expressing OneProt, TetR(+LVA), TetR(no LVA), wild-type and odor-free E. coli YYC912
 
 
<br>
 
<br>
 +
By comparing the growth curve of ''E. coli'' K12 expressing OneProt, TetR(+LVA) and TetR(no LVA) it is seen that the growth-rate of ''E. coli'' expressing TetR is lowered compared to the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. Loweing the levels of TetR would probably alleviate the attenuated growth-rate.
 
<br>
 
<br>
−
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed Caenorhabditis elegans (C. elegans) with E. coli K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. On both plates, 20 C. elegans were tested. Articles recommend using heat chock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, repeated. [1][2]
+
[[File:2014SDUGrowth curve (WT, OneProt, Empty vector).png]]
 
<br>
 
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 +
Growth curve illustrating the growth of ''E. coli'' K12 MG1655 WT, ''E. coli'' K12 expressing OneProt in pSB1C3 and with an empty vector pSB1C3.
 
<br>
 
<br>
−
After approximately 5 hours still no effects on C. elegans was detectable. Therefore we decided to stress C. elegans a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 hours, every C. elegans on both plates were alive. Thus we conclude that the protein has no toxic effect.
+
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed ''Caenorhabditis elegans'' (''C. elegans'') with ''E. coli'' K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. On both plates, 20 C. elegans were tested. Articles recommend using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, repeated. [1][2]
 
<br>
 
<br>
−
[[File:2014SDUC elegans.jpg|300px|thumb|left|Picture of C. elegans fed with E. coli K12 MG1655 expressing OneProt.]]
 
 
<br>
 
<br>
 +
After approximately 5 hours no effect on ''C. elegans'' was detectable. Therefore we decided to stress ''C. elegans'' a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 hours, every ''C. elegans''both plates were alive. Thus we conclude that the strain expressing OneProt at present levels doesn't display toxic effects on ''C. elegans''. This suggest that there are no obvious toxic effects towards eukaryotic cells such as human cells. However, the results are far from conclusive, and extensive testing is necessary to confirm this suggestion.
 +
<br>
 +
[[File:2014SDUC elegans.jpg|300px|thumb|left|Picture of ''C.elegans'' fed with ''E. coli'' K12 MG1655 expressing OneProt.]]
 
<br>
 
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−
 
−
===References===
 
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1. Mosbech, M., et al.: Functional Loss of Two Ceramide Synthases Elicits Autophagy-Dependent Lifespan Extension in C. elegans.: PLoS ONE, 2013. 8 vol:7. (http://www.ncbi.nlm.nih.gov/pubmed/23894595
 
 
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2. Rodriguez, M., et al.:Worms under stress: C. elegans sterss response and its relevance to complex human disease and aging. Trends in Genetics, 2013. Vol: 29, 6, p. 367-374. http://www.sciencedirect.com/science/article/pii/S016895251300022X
 
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 22:43, 25 October 2014

Protein containing all amino acids

Protein containing all the essential amino acids in the right ratio, accordig to WHO (see references) and also containing all the non essential amino acids.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

The protein is self-designed, so we wanted to test if the protein was expressed in E. coli K12 MG1655, by the use of Western blotting. The Western blot was blottet with E. coli K12 MG1655 wild-type and E. coli expressing OneProt at different OD600 measures.

Western blot showing E. coli wild-type at OD600 at 0.3, 0.8 and 1.8 and E. coli expressing OneProt at OD600 at 0.3, 0.8, 1.8 and an overnight culture.














The protein has a 3xFLAG tag and since bonds are showing under incubation with anti-FLAG, OneProt is expressed. However, from this Western blot, we cannot see if the protein has been cleaved, just that it is expressed.

In order to check that we had the protein expressed in its full length, we did a coomassie stain on a SDS-PAGE. Here we also wanted to receive information on the expression of the protein at different growth stages of E. coli. We analyzed samples from early exponential phase (OD600=0.3), late exponential phase (OD600=1.5), stationary phase (OD=2.5) and an overnight culture. As a control, E. coli with an empty vector (PSC1C3) was used.
Coomassie oneprot.PNG
OneProt has a molecular weight of approximately 53.7 kDa. Unfortunately, there is no clear bond at this length. However, there is a bond at approximately 25 kDa, which is not detected in the control. We cannot conclude what gives rise to the band, but it might be a cellular response to an unfolded protein.

By comparing the growth curve of E. coli K12 expressing OneProt, TetR(+LVA) and TetR(no LVA) it is seen that the growth-rate of E. coli expressing TetR is lowered compared to the other strains, which means that it might be difficult to have OneProt expressed in high amounts controlled by pTet (+/-LVA), with the TetR repressors overexpressed at present levels. Loweing the levels of TetR would probably alleviate the attenuated growth-rate.
2014SDUGrowth curve (WT, OneProt, Empty vector).png
Growth curve illustrating the growth of E. coli K12 MG1655 WT, E. coli K12 expressing OneProt in pSB1C3 and with an empty vector pSB1C3.
Because OneProt is self-designed, we wanted to test if the protein has any toxicity. To do so, we fed Caenorhabditis elegans (C. elegans) with E. coli K12 MG1655 containing an empty vector and a vector expressing OneProt on separate plates. On both plates, 20 C. elegans were tested. Articles recommend using heat shock assay for 7 hours: 1 hour at 35° C followed by 1 hour at 22°C, repeated. [1][2]

After approximately 5 hours no effect on C. elegans was detectable. Therefore we decided to stress C. elegans a little more, incubating them in 2 hours at 35°C followed by 1 hour at 22°C, repeated. After 7 hours, every C. elegansboth plates were alive. Thus we conclude that the strain expressing OneProt at present levels doesn't display toxic effects on C. elegans. This suggest that there are no obvious toxic effects towards eukaryotic cells such as human cells. However, the results are far from conclusive, and extensive testing is necessary to confirm this suggestion.

Picture of C.elegans fed with E. coli K12 MG1655 expressing OneProt.