Team:SDU-Denmark/project-p

From 2010.igem.org

(Difference between revisions)
(References)
(Characterization of parts)
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== K343004 ==
 
-
The FlhDC operon is the master regulator of flagella synthesis. A more detailed description of the operon can be found [https://2010.igem.org/Team:SDU-Denmark/project-t#Hyperflagellation here] <br>
 
-
In our system the purpose of the composite part is to hyper flagellate our cells so that a grater force can be generated in the microtubes. The FlhDC operon is naturally found in the ''E. coli'' strain MG1655 genome. We extracted the operon and inserted a silent mutation (T to C) at position 822 in the operon because without the mutation this site is a Pst1 digestion site and it would therefor constitute problems when assembling the composit part. <br> We have made three FlhDC parts:<br> [http://partsregistry.org/Part:BBa_K343100 K343100] is the coding sequence of the native FlhDC operon with the Pst1 digestion site <br> [http://partsregistry.org/Part:BBa_K343000 K343000] is the coding sequence of the mutated FlhDC operon <br> [http://partsregistry.org/Part:BBa_K343004 K343004] is the composite part containing the TetR repressable promoter (constitutive when no TetR is pressent) + RBS (J13002), the K343000 part and the double terminator (B0015). <br>
 
-
The composite part is caracterized firstly by using a motility assay and secondly by measuring plasmid stability and cell growth. <br><br>
 
-
 
-
===Motility assay===
 
-
The purpose of this experiment is to test the motility of the transformed cells containing either pSB1C3-K343004 or pSB3K3-K343004. We also want to test if it makes a difference in the motility whether the bacteria contain low-medium- or high-copy plasmids. <br><br>
 
-
For the motility assays we added 5ul of an ON culture to petridishes containing motility agar (LB media with 0.3% agar) instead of regular LA (Luria agar). This semi-solid media lets the bacteria swim more easily. <br>
 
-
In the assays three control plates were made: A negative control containing ''E. coli'' strain '''DH5alpha''' which does not express flagella and therefore movement in the media should be minimal.  A positive control containing ''E. coli'' strain H10407 which is a hyper flagellated class II pathogen, these bacteria should show high motility. And a wild type ''E. coli'' strain '''MG1655''' that has about 4 flagella per cell these cells would be expected to move farther than the DH5alpha but not as far as the positive control. <br><br>
 
-
The plates were incubated at 37 degrees celcius for up to 48 hours. <br>
 
-
The assay was carried out three times. All three times pictures were taken after 24 hours and in two of the assays pictures were also taken after 48 hours. In two of the assays 8.5 cm petridishes was used, the purpose of these assays was to se if the motility of our transformants differed from the control strains. In the third assay we used 13.5 cm petridishes to se ''how far'' our transformants were able to swim compared to the wild type ''E. coli'' strain MG1655 since the motility of the transformed cells seem to surpass the size of the 8.5 cm petri dishes. <br><br>
 
-
All pictures taken after 24 hours are shown in the last part of this section while the pictures taken after 48 hours are shown and described. <br>
 
-
''' WT, negative- and positive-control after 48 hours:''' <br>
 
-
[[Image:Team SDU-Denmark motility exp 3 DH5alpha.JPG|150px|DH5alpha after 48 hours]]
 
-
[[Image:Team SDU-Denmark motility exp 3 MG1655.JPG|150px|MG1655 after 48 hours]]
 
-
[[Image:Team SDU-Denmark motility exp 3 H10407.JPG|150px|H10407 after 48 hours]] <br><br>
 
-
[[Image:Team SDU-Denmark motility exp 3 pSB1C3-K343004.JPG|300px|FlhDCmut CP in pSB1C3 after 48 hours]]
 
-
[[Image:Team SDU-Denmark motility exp 3 pSB3K3-K343004.JPG|300px|FlhDCmut CP inpSB3K3 after 48 hours]]<br><br>
 
-
In all three assays we saw that the control plates that contain no antibiotics are contaminated with other bacterial colonies. We also see that the negative control has low motility, but they are not immotile. After 24 hours the negative control has not moved much but after 48 hours the length from center to edge of the colony is 1.5cm. The wild type has moved a bit farther than the negative control after 24 hours, and after 48 hours the difference from center to edge of the wild type is 2.5cm. The positive control moved about as far in 24 hours as the wild type did in 48 which and after 48 hours it had moved to the edges of the petridish (4.25cm). <br><br>
 
-
The ''E. Coli'' strain MG1655 cells transformed with pSB1C3-K343004 shows that after 24 hours these bacteria have moved which is farther than the wild type and the negative control. The ''E. coli'' strain MG1655/pSB3K3-K343004 have moved farthest of all 5 cultures after 24 hours. <br>
 
-
A difference in the way the two transformants move are seen in all three assays. The cells containing the pSB3K3 plasmid show a uniform circle whereas the cells transformed with the pSB1C3 plasmid shows a budding-pattern spreading from the center. After 48 hours the pSB1C3 cells are still not covering the entire plate. <br><br>
 
-
'''''Pictures of plates after 24 hours''''' <br>
 
-
'''Experiment 1.''' <br>
 
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-DH5a.JPG|100px|DH5alpha after 24 hours]]
 
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-MG1655.JPG|100px|MG1655 after 24 hours]]
 
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB1C3_(LA+chlor).JPG|100px|pSB1C3-FlhDCmutCP after 24 hours.]]
 
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB3K3_(LA+kan).JPG|100px|pSB3K3-FlhDCmutCP after 24 hours]]<br><br>
 
-
'''Experiment 2.''' <br>
 
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp2-DH5a.JPG|100px|DH5alpha after 24 hours]]
 
-
[[Image:Team-SDU-Denmark-Flagellamotility-exp2-MG1655.JPG|100px|MG1655 after 24 hours]]
 
-
[[Image:Team SDU-Denmark motility exp 2 FlhDCmutCP in pSB1C3.JPG|100px|FlhDCmutCP in pSB1C3 after 24 hours]]
 
-
[[Image:Team SDU-Denmark motility exp 2 FlhDCmutCP in pSB3K3.JPG|100px|FlhDCmutCP in pSB3K3 after 24 hours]]
 
-
[[Image:Team SDU-Denmark exp 2 H10407.JPG|100px|H10407 after 24 hours]]<br><br>
 
-
 
-
''' In conclusion''' <br>
 
-
From the pictures above we can definately se that the bacteria containing our part is much more motile than the wild type and the negative control. We assume this is caused by overexpression of the FlhDC master flagella operon which leads to hyperflagellation of the cells. <br>
 
-
The pictures taken after 24 hours show that bacteria with pSB1C3-K343004 have not moved as far as the bacteria containing pSB3K3-K343004. pSB1C3 is a high copy plasmid while pSB3K3 is a low-medium copy plasmid. The promoter in K343004 is a constitutive promoter (tetR repressable promoter). Bacteria containing a high copy plasmid with a constitutive promoter are more metabolically challanged than bacteria containing a low- or medium-copy plasmid with a constitutive promoter because of the higher number of plasmids per the cell. Therefore the high copy plasmid bacteria move slower than low- or medium-copy plasmid bacteria. However, after 48 hours there was only little difference in the spread. <br><br>
 
-
 
-
===Flagella staining===
 
-
<br>
 
-
When the ''E.coli'' strain MG1655 have a constitutive active transcription of the FlhDC operon more flagella’s is expected. The idea was to get a quantitative measurement of the increased flagella production by comparing the observed number flagellas on the wildtype MG1655 and the hyperflagellated MG1655. We tried to stain the flagella using silver staining and afterwards examined the bacteria under the microscope. We started the staining procedure before we had the mutated FlhDC operon as a composite part because we then had time to optimize the staining protocol and become really good at it. We started with staining of DH5α and H10407;  A ''E.coli'' strain which do not express flagellas (negative controle) and a hyperflagellated ''E.coli'' (positive control), respectively. The bacteria were grown on agar plates ON and stained with this protocol [https://2010.igem.org/Team:SDU-Denmark/protocols#FS1.2 FS1.2]. <br> <br>
 
-
Unfortunately it was not possible to see a clear and significant difference between the positive and negative control. Though at some placeses it could look like flagellas on the positive control but it was never enough to determine a difference. After repeating the protocol four times, each time with multible samples, we decided to reject this method, and another approach for characterizing our bio brick was employed. <br>
 
-
 
-
[[Image:Team_SDU-Denmark_Silver_staining_of_H10407_a_positive_control..JPG|300px|Silver staining of H10407: a positive control.]][[Image:Team_SDU-Denmark_Silver_staining_of_DH5α_the_negative_control.JPG‎|300px|Silver staining of DH5alpha: a negative control]]
 
-
 
-
=== Scanning Electron microscopy ===
 
-
<br>
 
-
=== Scanning Electron microscopy ===
 
-
<br>
 
-
After the failed staning of flagellas we tried to visualize the flagellas with scanning electron microscopy (SEM). The bacteria were grown ON in liquid cultures (5 ml LB-media). The bacteria were diluted to approximately 106 cells pr 10 µl solution. At the time being we did not have our FlhDC as a composite part so therefore we only tried SEM with the negative control strains DH5alpa and MG1655. We did this as a preliminary work so we were ready to do microscopy on the MG1655 containing the composite part when it is ready. Unfortunately the limited resolution and magnitude of the SEM at our disposal made it practically impossible to visualize any flagella in the microscope. Thus if this approach is to be used for characterization, a SEM of higher magnitude and resolution is required.<br>
 
-
The pictures below are SEM pictures the negative control strains DH5alpha and MG1655.<br>
 
-
 
-
[[Image:Team SDU-Denmark_DH5alpha_MAG_5.0_kx_and_EHT_7.5_kV.JPG|300px|SEM picture of DH5alpa a negative control]][[Image:Team_SDU-Denmark_Mg1655_MAG5kx_and_EHT_7.5kV.JPG|300px|SEM picture of MG1655 a negative control ]]
 
-
 
-
===Stability assay===
 
-
To dertermine the stability of our pSB1C3-K343004 plasmid, a stability experiment was carried out according to protocol[[https://2010.igem.org/Team:SDU-Denmark/protocols#Stability_assay SA1.1]]. ''E.coli'' MG1655/pSB1C3-K343004 was grown in LB media without chloramphenicol, whereby no selection pressure is excerted on the bacteria. Dilutions of the culture was spreaded on LA plates and LA plates with 35ug/mL chloramphenicol, respectively, and the colony forming units (cfu) was determined for each plate. The cfu for the LA plates represents the total amount of bacteria in the culture, and the cfu of LA plates with chloraphenicol corresponds to the amount of plasmid carrying bacteria. The percentage of the total amount of bacteria carrying the plasmid was plotted in a semi-logarithmic graph as a function of number of generation.
 
-
As seen in the graph, almost all of the bacteria had shedded the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. Thereby when the bacteria are carrying a high-copy plasmid like pSB1C3-K343004 it is to expect that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure.
 
-
It is likely to believe that pSB3C5-K343007, since being a low-copy plasmid, will not excert as much strain on the bacteria, and might therefore be stable for more genrations than pSB1C3-K343007. Therefore a stability assay of this plasmid might be of interest.
 
-
 
-
===Growth rate assay===
 
-
The purpose of this assay is to see if our transformants deviate from the wild type in growth rate. In the growth measurement assay we have measured OD at 550 nm every hour for 12 hours and at hour 24. In the experimental setup we used no lag phase was observed in any of the measurments. <br> The graph below shows the growth of our wild type ''E. coli'' strain MG1655, the MG1655 transformed with K343004 in pSB3K3 and in pSB1C3 respectively. <br><br> <br><br>
 
-
[[Image:Team SDU-Denmark OD WT+FlhDCmutCP.JPG|400px|OD of WT+FlhDCmutCP]] <br> [https://static.igem.org/mediawiki/2010/c/c5/Team_SDU-Denmark_Growth_rate_assay_FlhDCmut_CP.zip Raw data]<br><br>
 
-
From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite conteradictory to our results obtained from the stability assay. The transitory stability of pSB1C3-K343004 suggests that it is highly unfavorable for the bacteria, wherefore it might be expected that the growth raate of the bacteria containg this plasmid would be affected. Thus, however much a disadvantage the plasmid pose to the bacteria, their growth rates are not significantly influenced by the plasmid.
 
== K343007 ==
== K343007 ==
Line 138: Line 75:
The purpose of this assay is to see if our transformants deviate from the wild type in growth rate. In the growth measurement assay we have measured OD at 550 nm every hour for 12 hours and at hour 24. In the experimental setup we used no lag phase was observed in any of the measurments. <br> The graph below shows the growth of our wild type ''E. coli'' strain MG1655, the MG1655 transformed with K343007 in pSB3T5 and in pSB1C3 respectively. <br><br>  
The purpose of this assay is to see if our transformants deviate from the wild type in growth rate. In the growth measurement assay we have measured OD at 550 nm every hour for 12 hours and at hour 24. In the experimental setup we used no lag phase was observed in any of the measurments. <br> The graph below shows the growth of our wild type ''E. coli'' strain MG1655, the MG1655 transformed with K343007 in pSB3T5 and in pSB1C3 respectively. <br><br>  
[[Image:Team SDU-Denmark OD WT+PS.JPG|400px]] <br>[https://static.igem.org/mediawiki/2010/2/21/Team_SDU_Denmark_Growth_rate_assay_2_PS.zip Raw data] <br><br>
[[Image:Team SDU-Denmark OD WT+PS.JPG|400px]] <br>[https://static.igem.org/mediawiki/2010/2/21/Team_SDU_Denmark_Growth_rate_assay_2_PS.zip Raw data] <br><br>
-
From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite conteradictory to our results obtained from the stability assay. The transitory stability of pSB1C3-K343007 suggests that it is highly unfavorable for the bacteria, wherefore it might be expected that the growth raate of the bacteria containg this plasmid would be affected. Thus, however much a disadvantage the plasmid pose to the bacteria, their growth rates are not significantly influenced by the plasmid.  
+
From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite conteradictory to our results obtained from the stability assay. The transitory stability of pSB1C3-K343007 suggests that it is highly unfavorable for the bacteria, wherefore it might be expected that the growth raate of the bacteria containg this plasmid would be affected. Thus, however much a disadvantage the plasmid pose to the bacteria, their growth rates are not significantly influenced by the plasmid. <br><br>
 +
 
 +
== K343004 ==
 +
The FlhDC operon is the master regulator of flagella synthesis. A more detailed description of the operon can be found [https://2010.igem.org/Team:SDU-Denmark/project-t#Hyperflagellation here] <br>
 +
In our system the purpose of the composite part is to hyper flagellate our cells so that a grater force can be generated in the microtubes. The FlhDC operon is naturally found in the ''E. coli'' strain MG1655 genome. We extracted the operon and inserted a silent mutation (T to C) at position 822 in the operon because without the mutation this site is a Pst1 digestion site and it would therefor constitute problems when assembling the composit part. <br> We have made three FlhDC parts:<br> [http://partsregistry.org/Part:BBa_K343100 K343100] is the coding sequence of the native FlhDC operon with the Pst1 digestion site <br> [http://partsregistry.org/Part:BBa_K343000 K343000] is the coding sequence of the mutated FlhDC operon <br> [http://partsregistry.org/Part:BBa_K343004 K343004] is the composite part containing the TetR repressable promoter (constitutive when no TetR is pressent) + RBS (J13002), the K343000 part and the double terminator (B0015). <br>
 +
The composite part is caracterized firstly by using a motility assay and secondly by measuring plasmid stability and cell growth. <br><br>
 +
 
 +
===Motility assay===
 +
The purpose of this experiment is to test the motility of the transformed cells containing either pSB1C3-K343004 or pSB3K3-K343004. We also want to test if it makes a difference in the motility whether the bacteria contain low-medium- or high-copy plasmids. <br><br>
 +
For the motility assays we added 5ul of an ON culture to petridishes containing motility agar (LB media with 0.3% agar) instead of regular LA (Luria agar). This semi-solid media lets the bacteria swim more easily. <br>
 +
In the assays three control plates were made: A negative control containing ''E. coli'' strain '''DH5alpha''' which does not express flagella and therefore movement in the media should be minimal.  A positive control containing ''E. coli'' strain H10407 which is a hyper flagellated class II pathogen, these bacteria should show high motility. And a wild type ''E. coli'' strain '''MG1655''' that has about 4 flagella per cell these cells would be expected to move farther than the DH5alpha but not as far as the positive control. <br><br>
 +
The plates were incubated at 37 degrees celcius for up to 48 hours. <br>
 +
The assay was carried out three times. All three times pictures were taken after 24 hours and in two of the assays pictures were also taken after 48 hours. In two of the assays 8.5 cm petridishes was used, the purpose of these assays was to se if the motility of our transformants differed from the control strains. In the third assay we used 13.5 cm petridishes to se ''how far'' our transformants were able to swim compared to the wild type ''E. coli'' strain MG1655 since the motility of the transformed cells seem to surpass the size of the 8.5 cm petri dishes. <br><br>
 +
All pictures taken after 24 hours are shown in the last part of this section while the pictures taken after 48 hours are shown and described. <br>
 +
''' WT, negative- and positive-control after 48 hours:''' <br>
 +
[[Image:Team SDU-Denmark motility exp 3 DH5alpha.JPG|150px|DH5alpha after 48 hours]]
 +
[[Image:Team SDU-Denmark motility exp 3 MG1655.JPG|150px|MG1655 after 48 hours]]
 +
[[Image:Team SDU-Denmark motility exp 3 H10407.JPG|150px|H10407 after 48 hours]] <br><br>
 +
[[Image:Team SDU-Denmark motility exp 3 pSB1C3-K343004.JPG|300px|FlhDCmut CP in pSB1C3 after 48 hours]]
 +
[[Image:Team SDU-Denmark motility exp 3 pSB3K3-K343004.JPG|300px|FlhDCmut CP inpSB3K3 after 48 hours]]<br><br>
 +
In all three assays we saw that the control plates that contain no antibiotics are contaminated with other bacterial colonies. We also see that the negative control has low motility, but they are not immotile. After 24 hours the negative control has not moved much but after 48 hours the length from center to edge of the colony is 1.5cm. The wild type has moved a bit farther than the negative control after 24 hours, and after 48 hours the difference from center to edge of the wild type is 2.5cm. The positive control moved about as far in 24 hours as the wild type did in 48 which and after 48 hours it had moved to the edges of the petridish (4.25cm). <br><br>
 +
The ''E. Coli'' strain MG1655 cells transformed with pSB1C3-K343004 shows that after 24 hours these bacteria have moved which is farther than the wild type and the negative control. The ''E. coli'' strain MG1655/pSB3K3-K343004 have moved farthest of all 5 cultures after 24 hours. <br>
 +
A difference in the way the two transformants move are seen in all three assays. The cells containing the pSB3K3 plasmid show a uniform circle whereas the cells transformed with the pSB1C3 plasmid shows a budding-pattern spreading from the center. After 48 hours the pSB1C3 cells are still not covering the entire plate. <br><br>
 +
'''''Pictures of plates after 24 hours''''' <br>
 +
'''Experiment 1.''' <br>
 +
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-DH5a.JPG|100px|DH5alpha after 24 hours]]
 +
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-MG1655.JPG|100px|MG1655 after 24 hours]]
 +
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB1C3_(LA+chlor).JPG|100px|pSB1C3-FlhDCmutCP after 24 hours.]]
 +
[[Image:Team-SDU-Denmark-Flagellamotility-exp1-FlhDCmutCP_i_pSB3K3_(LA+kan).JPG|100px|pSB3K3-FlhDCmutCP after 24 hours]]<br><br>
 +
'''Experiment 2.''' <br>
 +
[[Image:Team-SDU-Denmark-Flagellamotility-exp2-DH5a.JPG|100px|DH5alpha after 24 hours]]
 +
[[Image:Team-SDU-Denmark-Flagellamotility-exp2-MG1655.JPG|100px|MG1655 after 24 hours]]
 +
[[Image:Team SDU-Denmark motility exp 2 FlhDCmutCP in pSB1C3.JPG|100px|FlhDCmutCP in pSB1C3 after 24 hours]]
 +
[[Image:Team SDU-Denmark motility exp 2 FlhDCmutCP in pSB3K3.JPG|100px|FlhDCmutCP in pSB3K3 after 24 hours]]
 +
[[Image:Team SDU-Denmark exp 2 H10407.JPG|100px|H10407 after 24 hours]]<br><br>
 +
 
 +
''' In conclusion''' <br>
 +
From the pictures above we can definately se that the bacteria containing our part is much more motile than the wild type and the negative control. We assume this is caused by overexpression of the FlhDC master flagella operon which leads to hyperflagellation of the cells. <br>
 +
The pictures taken after 24 hours show that bacteria with pSB1C3-K343004 have not moved as far as the bacteria containing pSB3K3-K343004. pSB1C3 is a high copy plasmid while pSB3K3 is a low-medium copy plasmid. The promoter in K343004 is a constitutive promoter (tetR repressable promoter). Bacteria containing a high copy plasmid with a constitutive promoter are more metabolically challanged than bacteria containing a low- or medium-copy plasmid with a constitutive promoter because of the higher number of plasmids per the cell. Therefore the high copy plasmid bacteria move slower than low- or medium-copy plasmid bacteria. However, after 48 hours there was only little difference in the spread. <br><br>
 +
 
 +
===Flagella staining===
 +
<br>
 +
When the ''E.coli'' strain MG1655 have a constitutive active transcription of the FlhDC operon more flagella’s is expected. The idea was to get a quantitative measurement of the increased flagella production by comparing the observed number flagellas on the wildtype MG1655 and the hyperflagellated MG1655. We tried to stain the flagella using silver staining and afterwards examined the bacteria under the microscope. We started the staining procedure before we had the mutated FlhDC operon as a composite part because we then had time to optimize the staining protocol and become really good at it. We started with staining of DH5α and H10407;  A ''E.coli'' strain which do not express flagellas (negative controle) and a hyperflagellated ''E.coli'' (positive control), respectively. The bacteria were grown on agar plates ON and stained with this protocol [https://2010.igem.org/Team:SDU-Denmark/protocols#FS1.2 FS1.2]. <br> <br>
 +
Unfortunately it was not possible to see a clear and significant difference between the positive and negative control. Though at some placeses it could look like flagellas on the positive control but it was never enough to determine a difference. After repeating the protocol four times, each time with multible samples, we decided to reject this method, and another approach for characterizing our bio brick was employed. <br>
 +
 
 +
[[Image:Team_SDU-Denmark_Silver_staining_of_H10407_a_positive_control..JPG|300px|Silver staining of H10407: a positive control.]][[Image:Team_SDU-Denmark_Silver_staining_of_DH5α_the_negative_control.JPG‎|300px|Silver staining of DH5alpha: a negative control]]
 +
 
 +
=== Scanning Electron microscopy ===
 +
<br>
 +
=== Scanning Electron microscopy ===
 +
<br>
 +
After the failed staning of flagellas we tried to visualize the flagellas with scanning electron microscopy (SEM). The bacteria were grown ON in liquid cultures (5 ml LB-media). The bacteria were diluted to approximately 106 cells pr 10 µl solution. At the time being we did not have our FlhDC as a composite part so therefore we only tried SEM with the negative control strains DH5alpa and MG1655. We did this as a preliminary work so we were ready to do microscopy on the MG1655 containing the composite part when it is ready. Unfortunately the limited resolution and magnitude of the SEM at our disposal made it practically impossible to visualize any flagella in the microscope. Thus if this approach is to be used for characterization, a SEM of higher magnitude and resolution is required.<br>
 +
The pictures below are SEM pictures the negative control strains DH5alpha and MG1655.<br>
 +
 
 +
[[Image:Team SDU-Denmark_DH5alpha_MAG_5.0_kx_and_EHT_7.5_kV.JPG|300px|SEM picture of DH5alpa a negative control]][[Image:Team_SDU-Denmark_Mg1655_MAG5kx_and_EHT_7.5kV.JPG|300px|SEM picture of MG1655 a negative control ]]
 +
 
 +
===Stability assay===
 +
To dertermine the stability of our pSB1C3-K343004 plasmid, a stability experiment was carried out according to protocol[[https://2010.igem.org/Team:SDU-Denmark/protocols#Stability_assay SA1.1]]. ''E.coli'' MG1655/pSB1C3-K343004 was grown in LB media without chloramphenicol, whereby no selection pressure is excerted on the bacteria. Dilutions of the culture was spreaded on LA plates and LA plates with 35ug/mL chloramphenicol, respectively, and the colony forming units (cfu) was determined for each plate. The cfu for the LA plates represents the total amount of bacteria in the culture, and the cfu of LA plates with chloraphenicol corresponds to the amount of plasmid carrying bacteria. The percentage of the total amount of bacteria carrying the plasmid was plotted in a semi-logarithmic graph as a function of number of generation.
 +
As seen in the graph, almost all of the bacteria had shedded the plasmid after 20 generations, suggesting that the plasmid is only stable within the cell for a few generations (<20). This is presumably due to the strain brought upon the bacteria by the plasmid. Thereby when the bacteria are carrying a high-copy plasmid like pSB1C3-K343004 it is to expect that the bacteria will quickly shed the plasmid when no longer exposed to a selection pressure.
 +
It is likely to believe that pSB3C5-K343007, since being a low-copy plasmid, will not excert as much strain on the bacteria, and might therefore be stable for more genrations than pSB1C3-K343007. Therefore a stability assay of this plasmid might be of interest.
 +
 
 +
===Growth rate assay===
 +
The purpose of this assay is to see if our transformants deviate from the wild type in growth rate. In the growth measurement assay we have measured OD at 550 nm every hour for 12 hours and at hour 24. In the experimental setup we used no lag phase was observed in any of the measurments. <br> The graph below shows the growth of our wild type ''E. coli'' strain MG1655, the MG1655 transformed with K343004 in pSB3K3 and in pSB1C3 respectively. <br><br> <br><br>
 +
[[Image:Team SDU-Denmark OD WT+FlhDCmutCP.JPG|400px|OD of WT+FlhDCmutCP]] <br> [https://static.igem.org/mediawiki/2010/c/c5/Team_SDU-Denmark_Growth_rate_assay_FlhDCmut_CP.zip Raw data]<br><br>
 +
From our data we see no significant difference between the plasmid carrying bacteria and the wild type. This can be said to be quite conteradictory to our results obtained from the stability assay. The transitory stability of pSB1C3-K343004 suggests that it is highly unfavorable for the bacteria, wherefore it might be expected that the growth raate of the bacteria containg this plasmid would be affected. Thus, however much a disadvantage the plasmid pose to the bacteria, their growth rates are not significantly influenced by the plasmid.
== K343005 ==  
== K343005 ==  

Revision as of 12:52, 26 October 2010