Difference between revisions of "Part:BBa K346004:Experience"

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how you used this part and how it worked out.
 
how you used this part and how it worked out.
  
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===Construction of BBa_K346004===
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===User Reviews===
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[[Image:mbp4.jpg]]
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<partinfo>BBa_R0051 AddReview 5</partinfo>
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<I> UFAM_Brazil 2014 </I>
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We used this biobrick part to build a Hg bioacumulator with [https://parts.igem.org/Part:BBa_K1355001 bidirectional promoter regulated by MerR]! This part worked so well, we have incredible results! Thanks, Peking!
  
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Just as what we have done to construct the mercury Metal Binding Peptide, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix. The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig), which was verified by DNA sequencing.Then the RBS and terminator is added.
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The graph represented on Figure 1 shows the amount of Hg in supernatant (LM medium recovered) and in [http://2014.igem.org/Team:UFAM_Brazil Mercury Bacter] (DH5-alpha transformed with [https://parts.igem.org/Part:BBa_K1355003 BBa_K1355003] at the time 1 (01:30 hours of bacteria incubation) and time two (03:00 hours of bacteria incubation); 
  
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Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – mbp , which was verified by DNA sequencing.
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[[File:bc6.png]]
  
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'''Figure 6:''' Metal binding peptide activity at 500 ppb in time 1 and 2
  
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==Expression Experiment and Function Test: ==
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It can be observed that the amount of Hg in Mercury Bacter bioaccumulator, increases according to the time of incubation. In the 500 ppb Hg concentration the control bacterium just accumulated 4% of total Hg amount. Instead, Mercury Bacter accumulated 58% of total Hg amount in just 03:00 hours of incubation!!! This is so awesome! 
  
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To test the function of this part, both expression experiment and function test is necessary. We have verified the size of the expressed proteins with SDS-page and Western blot. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES with the mercury gradient from 10^-8M to 10^-6M.
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Check it out our [https://parts.igem.org/Part:BBa_K1355003:Experience Experience] in extended version and the [https://parts.igem.org/Part:BBa_K1355003:Design Desing] [http://2014.igem.org/Team:UFAM_Brazil Mercury Bacter] of the Hg biosensor!
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<!-- End of the user review template -->
  
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===Construction of BBa_K346004===
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[[Image:mbp4 v2.jpg]]
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Just as what we have done to construct the mercury Metal Binding Peptide, the entire coding region of the MBP for standard part was amplified by PCR from full length PbrR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in PbrR and was added to afford some flexibility in the loop connecting the two dimerization helix. The two PCR products were digested with EcoR I / BspEI, or BspEI / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 bybone step (Fig above), which was verified by DNA sequencing. Then the RBS and terminator was prefixed and suffixed.
 +
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Based on the similar method, MBP-His6 was constructed by using two different pairs of primers, which was used for western blotting. The two PCR products were digested with Nde I / BspEI, or BspEI / Xho I and cloned into Nde I / Xho I -digested pET 21a backbone which contains a six his tag downstream. Cloning was verified by DNA sequencing.
 +
 +
==Expression Experiment and Function Test: ==
 +
 +
To test the function of this part, both expression experiment and function test is necessary. We have verified the size of the expressed proteins with SDS-PAGE and Western blotting. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES
  
 
== Results: ==
 
== Results: ==
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'''Expression of proteins'''
 
'''Expression of proteins'''
  
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The plasmid PET21a-mbp is transferred to E.coli strain BL21, which can generate T7polyerase when induced with IPTG. Both induced cells and uninduced cells(as control) are centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-page and Western blot of the expressed proteins show that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD  which consists with the predicted size, indicating that the engineered MBP can be expressed in the cytosol.
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The plasmid PET21a-MBP was transformed to E.coli strain BL21. Both induced cells and uninduced cells(as control) were centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-PAGE and Western blotting of the expressed proteins showed that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD  as expected.
  
 
[[Image:results of MBP(Pb).jpg]]
 
[[Image:results of MBP(Pb).jpg]]
  
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The specific band in western blot for his-tag fused MBP of about 12 kD confirmed that the MBP is expressed as expected. Considerable amount of MBP expressed in cytosol can be indicated from the result of SDS-PAGE.
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The specific band in western blotting for his-tag fused MBP of about 12 kD confirmed that the MBP was expressed as expected. Considerable amount of MBP expressed in cytosol can be indicated from the result of SDS-PAGE.
  
  
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'''Function test'''
 
'''Function test'''
  
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Having made sure that the protein can express normally in the cytosol, the function tests experiment are carried out with ICP-AES. To test the efficiency of mercury absorption of MBP in different concentration of mercury, the concentration gradient of lead is set from 10^-8M to 10^-6M, the results are shown in figure as followed.
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Having confirmed that the protein can be expressed normally in the cytosol, We conducted the function tests experiment with ICP-AES. To test the efficiency of mercury absorption by MBP in different concentration of mercury, the concentration gradient of lead was set from 10^-7M to 10^-5M （data not shown). The results of the efficiency of different parts containing MBP(lead) was shown below. It is apparent that under the concentration of 10^-5M, there was a notable absorption of lead compared to the control.
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[[Image:Pb.png]]
  
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[[Image:Pb.jpg]]
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Figure 2 Lead binding capacity of lead absorbed by bacteria with MBP expressed in different subcellular compartments cultured for ~40h in 10-5 mol/L Pb (II) medium.
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Latest revision as of 23:58, 17 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

User Reviews

•••••

UFAM_Brazil 2014

We used this biobrick part to build a Hg bioacumulator with bidirectional promoter regulated by MerR! This part worked so well, we have incredible results! Thanks, Peking!

The graph represented on Figure 1 shows the amount of Hg in supernatant (LM medium recovered) and in [http://2014.igem.org/Team:UFAM_Brazil Mercury Bacter] (DH5-alpha transformed with BBa_K1355003 at the time 1 (01:30 hours of bacteria incubation) and time two (03:00 hours of bacteria incubation);

Bc6.png

Figure 6: Metal binding peptide activity at 500 ppb in time 1 and 2

It can be observed that the amount of Hg in Mercury Bacter bioaccumulator, increases according to the time of incubation. In the 500 ppb Hg concentration the control bacterium just accumulated 4% of total Hg amount. Instead, Mercury Bacter accumulated 58% of total Hg amount in just 03:00 hours of incubation!!! This is so awesome!

Check it out our Experience in extended version and the Desing [http://2014.igem.org/Team:UFAM_Brazil Mercury Bacter] of the Hg biosensor!



Construction of BBa_K346004

Mbp4 v2.jpg

Just as what we have done to construct the mercury Metal Binding Peptide, the entire coding region of the MBP for standard part was amplified by PCR from full length PbrR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in PbrR and was added to afford some flexibility in the loop connecting the two dimerization helix. The two PCR products were digested with EcoR I / BspEI, or BspEI / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 bybone step (Fig above), which was verified by DNA sequencing. Then the RBS and terminator was prefixed and suffixed.

Based on the similar method, MBP-His6 was constructed by using two different pairs of primers, which was used for western blotting. The two PCR products were digested with Nde I / BspEI, or BspEI / Xho I and cloned into Nde I / Xho I -digested pET 21a backbone which contains a six his tag downstream. Cloning was verified by DNA sequencing.

Expression Experiment and Function Test:

To test the function of this part, both expression experiment and function test is necessary. We have verified the size of the expressed proteins with SDS-PAGE and Western blotting. Besides, to test the efficiency of mercury binding, we also carried out the function test with ICP-AES

Results:

Expression of proteins

The plasmid PET21a-MBP was transformed to E.coli strain BL21. Both induced cells and uninduced cells(as control) were centrifuged to get the cytosol, the periplasm and the membrane separated. The SDS-PAGE and Western blotting of the expressed proteins showed that induced cells expressed an identical IPTG-inducible protein at the proper place with the size of ~12kD as expected.

Results of MBP(Pb).jpg

The specific band in western blotting for his-tag fused MBP of about 12 kD confirmed that the MBP was expressed as expected. Considerable amount of MBP expressed in cytosol can be indicated from the result of SDS-PAGE.



Function test

Having confirmed that the protein can be expressed normally in the cytosol, We conducted the function tests experiment with ICP-AES. To test the efficiency of mercury absorption by MBP in different concentration of mercury, the concentration gradient of lead was set from 10^-7M to 10^-5M （data not shown). The results of the efficiency of different parts containing MBP(lead) was shown below. It is apparent that under the concentration of 10^-5M, there was a notable absorption of lead compared to the control.

Pb.png

Figure 2 Lead binding capacity of lead absorbed by bacteria with MBP expressed in different subcellular compartments cultured for ~40h in 10-5 mol/L Pb (II) medium.



User Reviews

UNIQ8f3088cf72b69bd5-partinfo-00000001-QINU UNIQ8f3088cf72b69bd5-partinfo-00000002-QINU