Difference between revisions of "Part:BBa K4417018"
 
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<h1>Description</h1>
 
<h1>Description</h1>
  
−
This part was created by cloning the master plasmid (BBa_K4417017) with the coding sequence of ureEFG (BBa_K4417016). This plasmid retained the nature urease operon sequence under a novel cumate inducible promoter (BBa_K4417007). The Type IIS backbone also contains a strong RBS (BBa_K4417008) and an rrnB T1 terminator (BBa_K4417011).
+
This part was created by cloning the master plasmid (<partinfo>BBa_K4417017</partinfo>) with the coding sequence of ureEFG (<partinfo>BBa_K4417016</partinfo>). This plasmid retained the native urease operon sequence under a novel cumate inducible promoter (<partinfo>BBa_K4417007</partinfo>). The Type IIS backbone also contains a strong RBS (<partinfo>BBa_K4417008</partinfo>) and an rrnB T1 terminator (<partinfo>BBa_K4417011</partinfo>).
  
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[[File:Wkebfke.png|600px|thumb|center|'''Figure 1:'''Urease nature operon construct from Sporosarcina pasteurii.]]
+
[[File:Zjy12345.png|400px|thumb|center|'''Figure 1:'''Urease native operon sequence from ''Sporosarcina pasteurii''.]]
  
 
<h1>Usage and Biology</h1>
 
<h1>Usage and Biology</h1>
−
* This part can be transformed in both B. subtilis and E. coli.  
+
* This part can be transformed in both ''B. subtilis'' and ''E. coli''.  
 
* Inducer: p-isopropyl benzoate (cumate).  
 
* Inducer: p-isopropyl benzoate (cumate).  
 
* Cumate is non-toxic to the host. In our experiment, we induced the urease expression with 50 μM cumate.  
 
* Cumate is non-toxic to the host. In our experiment, we induced the urease expression with 50 μM cumate.  
−
* E. coli ori is a pMB1 derivative  
+
* ''E. coli'' ori is a pMB1 derivative  
−
* B. sub ori is unknown
+
* ''B. sub'' ori is unknown
−
* The copy number of this plasmid in B. subtilis and E. coli is unknown.
+
* The copy number of this plasmid in ''B. subtilis'' and ''E. coli'' is unknown.
−
* NdeI restriction can be used to change the promoter.  
+
* ''Nde''I and ''Sac''I restriction digest can be used to change the promoter.  
  
 
<h1>Cloning Strategy</h1>
 
<h1>Cloning Strategy</h1>
−
[[File:Oiho.png|600px|thumb|center|'''Figure 2:'''CuO-RBS-ureABC-ureEFG-rrnB T1 Terminator Nature Operon.]]
+
Golden Gate Assembly was used to assemble the master plasmid with TU2B.
 +
 
 +
[[File:Zjy987.png|600px|thumb|center|'''Figure 2:'''CuO-RBS-ureABC-ureEFG-rrnB T1 Terminator native operon sequence.]]
  
 
<h1>Method</h1>
 
<h1>Method</h1>
−
We used Golden Gate Assembly to clone the ureEFG part into the master plasmid, following NEeB’s Golden Gate Assembly Protocol (https://international.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600) using BsaI restriction site.  
+
We used Golden Gate Assembly to clone the ureEFG part into the master plasmid, following NEB’s Golden Gate Assembly Protocol (https://international.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600) using ''Bsa''I restriction site.  
  
 
Golden Gate Assembly protocol:  
 
Golden Gate Assembly protocol:  
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** 0.5 μL T4 DNA Ligase
 
** 0.5 μL T4 DNA Ligase
 
** 2 μL of 10X T4 Ligase buffer  
 
** 2 μL of 10X T4 Ligase buffer  
−
** 0.5 μL BsaI enzyme
+
** 0.5 μL ''Bsa''I enzyme
 
** 100ng of receiver plasmid
 
** 100ng of receiver plasmid
 
** Equimolar amounts of inserts
 
** Equimolar amounts of inserts
Line 41: Line 43:
  
  
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[[File:Uuuu.png|600px|thumb|center|'''Figure 3:'''E. coli plates transformed with urease nature operon plasmid. (a), (b) transformed with no bME. (c), (d) transformed with 24mM bME.]]
+
[[File:Uuuu.png|600px|thumb|center|'''Figure 3:'''''E. coli'' plates transformed with urease native operon plasmid. (a), (b) transformed with no bME. (c), (d) transformed with 24mM bME.]]
  
−
Figure 3 verified that bME could be used to improve the transformation efficiency to a certain extent. After successful transformation, colonies were picked, grown, and the plasmid isolated. The isolated plasmid was checked by SapI diagnostic digest. 5 μL uncut and 10 μL cut samples were loaded on a 1% agarose gel to check the band size. From Figure 4, expected bands can be observed around 6880bp and 4789bp.
+
Figure 3 verified that bME could be used to improve the transformation efficiency to a certain extent. After successful transformation, colonies were picked, grown, and the plasmid isolated. The isolated plasmid was checked by ''Sap''I diagnostic digest. 5 μL uncut and 10 μL cut samples were loaded on a 1% agarose gel to check the band size. From Figure 4, expected bands can be observed around 6880bp and 4789bp.
  
−
[[File:Adasd.png|600px|thumb|center|'''Figure 4:'''Restriction digest of urease nature operon plasmid. 1: DNA ladder, 2,4,6,8: nature operon plasmid cut with SapI, 3,5,7,9: nature operon plasmid uncut, 10: TU1 cut with SapI, 11: TU1 uncut.]]
+
[[File:Adasd.png|600px|thumb|center|'''Figure 4:'''Restriction digest of urease native operon plasmid. 1: DNA ladder, 2,4,6,8: nature operon plasmid cut with ''Sap''I, 3,5,7,9: nature operon plasmid uncut, 10: TU1 cut with ''Sap''I, 11: TU1 uncut.]]
  
 
<h1>Characterization</h1>
 
<h1>Characterization</h1>
  
−
In order to observe whether the ureABCEFG was successfully expressed, we analysed our cell pellet using SDS PAGE. The pellet obtained from the 10 mL cultures was then resuspended in Tris Buffer Saline at an OD600. Once resuspended, the sample was cell lysed using sonication. Following sonication, the samples were spanned to separate the soluble and insoluble fragments from the whole cell lysate. 60 μL from each sample were obtained and stained with Laemmli reagent.
+
In order to observe whether the ureABCEFG genes were successfully expressed in DH5-α, we analysed our the soluble cell lysate by SDS PAGE. The cell pellet obtained from the 10 mL culture was resuspended in Tris Buffer Saline. Once resuspended, the sample was lysed using sonication. Following sonication, the samples were centrifuged to separate the soluble and insoluble fractions from the whole cell lysate. 60 μL from each sample were taken and boiled for 10 min with Laemmli buffer to denature the sample.
  
−
[[File:Sdfsdfsfd.png|600px|thumb|center|'''Figure 5:'''SDS PAGE of full urease operon in E. coli. All the strains were grown in LB medium; 1: PageRuler Protein Ladder, 2: WT E. coli cell lysate, 3: WT E. coli soluble fragment, 4: SDM1,3 TU1 cell lysate, 5: SDM1,3 TU1 soluble fragment, 6: pCT5c TU1 cell lysate, 7: pCT5c TU1 soluble fragment, 8: SDM1,3 TU2 cell lysate, 9: SDM1,3 TU2 soluble fragment, 10: pCT5c TU2 cell lysate, 11: pCT5c TU2 soluble fragment, 12: pCT5c full urease operon cell lysate, 13: pCT5c full urease operon soluble fragment, 14: pCT5c full urease operon cell lysate, 15: pCT5c full urease operon soluble fragment. ]]
+
[[File:Sdfsdfsfd.png|600px|thumb|center|'''Figure 5:'''SDS PAGE of native urease operon in ''E. coli''. All the strains were grown in LB medium, and only the soluble fraction was loaded; 1: PageRuler Protein Ladder, 2: WT ''E. coli'' cell lysate, 3: WT ''E. coli'' soluble fragment, 4: SDM1,3 TU1 cell lysate, 5: SDM1,3 TU1 soluble fragment, 6: pCT5c TU1 cell lysate, 7: pCT5c TU1 soluble fragment, 8: SDM1,3 TU2 cell lysate, 9: SDM1,3 TU2 soluble fragment, 10: pCT5c TU2 cell lysate, 11: pCT5c TU2 soluble fragment, 12: pCT5c full urease operon cell lysate, 13: pCT5c full urease operon soluble fragment, 14: pCT5c native urease operon cell lysate, 15: pCT5c native urease operon soluble fragment. ]]
  
 
From Figure 5, it could be concluded that the urease was successfully produced since the correct bands are observed in both the soluble and insoluble fragment. ureC was identified at 61.5 kDa, and ureF was identified at 24.9 kDa.  
 
From Figure 5, it could be concluded that the urease was successfully produced since the correct bands are observed in both the soluble and insoluble fragment. ureC was identified at 61.5 kDa, and ureF was identified at 24.9 kDa.  

Latest revision as of 02:29, 14 October 2022


CuO-RBS-ureABC-ureEFG-rrnB T1 Native Operon Sequence

Description

This part was created by cloning the master plasmid (BBa_K4417017) with the coding sequence of ureEFG (BBa_K4417016). This plasmid retained the native urease operon sequence under a novel cumate inducible promoter (BBa_K4417007). The Type IIS backbone also contains a strong RBS (BBa_K4417008) and an rrnB T1 terminator (BBa_K4417011).

Figure 1:Urease native operon sequence from Sporosarcina pasteurii.

Usage and Biology

  • This part can be transformed in both B. subtilis and E. coli.
  • Inducer: p-isopropyl benzoate (cumate).
  • Cumate is non-toxic to the host. In our experiment, we induced the urease expression with 50 μM cumate.
  • E. coli ori is a pMB1 derivative
  • B. sub ori is unknown
  • The copy number of this plasmid in B. subtilis and E. coli is unknown.
  • NdeI and SacI restriction digest can be used to change the promoter.

Cloning Strategy

Golden Gate Assembly was used to assemble the master plasmid with TU2B.

Figure 2:CuO-RBS-ureABC-ureEFG-rrnB T1 Terminator native operon sequence.

Method

We used Golden Gate Assembly to clone the ureEFG part into the master plasmid, following NEB’s Golden Gate Assembly Protocol (https://international.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600) using BsaI restriction site.

Golden Gate Assembly protocol:

  • The following reagents were assembled in a thin-walled PCR tube.
    • 0.5 μL T4 DNA Ligase
    • 2 μL of 10X T4 Ligase buffer
    • 0.5 μL BsaI enzyme
    • 100ng of receiver plasmid
    • Equimolar amounts of inserts
    • MilliQ for a total volume of 20 μL
  • Mixed gently.
  • Set up the thermocycler program.


Hbhbhbh.png


Figure 3:E. coli plates transformed with urease native operon plasmid. (a), (b) transformed with no bME. (c), (d) transformed with 24mM bME.

Figure 3 verified that bME could be used to improve the transformation efficiency to a certain extent. After successful transformation, colonies were picked, grown, and the plasmid isolated. The isolated plasmid was checked by SapI diagnostic digest. 5 μL uncut and 10 μL cut samples were loaded on a 1% agarose gel to check the band size. From Figure 4, expected bands can be observed around 6880bp and 4789bp.

Figure 4:Restriction digest of urease native operon plasmid. 1: DNA ladder, 2,4,6,8: nature operon plasmid cut with SapI, 3,5,7,9: nature operon plasmid uncut, 10: TU1 cut with SapI, 11: TU1 uncut.

Characterization

In order to observe whether the ureABCEFG genes were successfully expressed in DH5-α, we analysed our the soluble cell lysate by SDS PAGE. The cell pellet obtained from the 10 mL culture was resuspended in Tris Buffer Saline. Once resuspended, the sample was lysed using sonication. Following sonication, the samples were centrifuged to separate the soluble and insoluble fractions from the whole cell lysate. 60 μL from each sample were taken and boiled for 10 min with Laemmli buffer to denature the sample.

Figure 5:SDS PAGE of native urease operon in E. coli. All the strains were grown in LB medium, and only the soluble fraction was loaded; 1: PageRuler Protein Ladder, 2: WT E. coli cell lysate, 3: WT E. coli soluble fragment, 4: SDM1,3 TU1 cell lysate, 5: SDM1,3 TU1 soluble fragment, 6: pCT5c TU1 cell lysate, 7: pCT5c TU1 soluble fragment, 8: SDM1,3 TU2 cell lysate, 9: SDM1,3 TU2 soluble fragment, 10: pCT5c TU2 cell lysate, 11: pCT5c TU2 soluble fragment, 12: pCT5c full urease operon cell lysate, 13: pCT5c full urease operon soluble fragment, 14: pCT5c native urease operon cell lysate, 15: pCT5c native urease operon soluble fragment.

From Figure 5, it could be concluded that the urease was successfully produced since the correct bands are observed in both the soluble and insoluble fragment. ureC was identified at 61.5 kDa, and ureF was identified at 24.9 kDa.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3592
    Illegal BamHI site found at 1
    Illegal XhoI site found at 4346
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]