Difference between revisions of "Part:BBa K2812002"

Latest revision as of 11:35, 17 October 2018

pBAD-ara promoter - Arabinose inducible regulatory promoter

Usage and Biology

The pBAD-ara promoter is part of the arabinose operon. A gene under the control of the pBAD-ara promoter can be induced by addition of arabinose. We as TU-Eindhoven 2018 used this promoter to control the release of the antimicrobials lysostaphin and pyocin.

Figure 1: SDS-PAGE gel of pBAD-ara promotor-Lysostaphin-thrombin-HlyA-His-tag construct protein expression experiment.

Protein Expression

Protein expression experiments were performed to characterize the functionality of the pBAD-ara promotor. To allow expression of the construct under the control of arabinose, it was assembled together with the BBa_K2812004 biobrick via Gibson assembly. After successful transformation into E. coli BL21 (DE3), the bacteria were cultured in LB medium at 37 °C. Prior to induction, a sample was taken (0) to establish the gene expression pattern of uninduced bacteria. One sample was induced at an OD600 of 0.5-0.8 by adding 2 mM arabinose to induce expression of the recombinant protein, also at 37 °C. Another sample was not induced as control. Samples were taken 4 hours after induction of both an induced (4+) and an uninduced culture (4-). SDS samples were prepared and loaded onto a polyacrylamide gel to yield the SDS-PAGE results that can be seen in figure 1.

Conclusion

The biobrick BBa_K2812004 should in theory result in the overexpression of a protein with a length of 50 kDa. However, as can be read in the extensive characterisation of BBa_K2812004, self-cleavage of the fusion protein is observed, generating a part with a mass of 23 kDa and a part with a mass of 27 kDa. In the induced sample, two bands slightly below and above 25 kDa can be observed, which are not present at all in the uninduced sample. This confirms the succesful induction of protein expression of the BBa_K2812004 construct, demonstrating that our pBAD-ara promotor is functional.

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]

@@ Line 2: / Line 2: @@
 __NOTOC__
 <partinfo>BBa_K2812002 short</partinfo>
+===Usage and Biology===
+The pBAD-ara promoter is part of the arabinose operon. A gene under the control of the pBAD-ara promoter can be induced by addition of arabinose. We as TU-Eindhoven 2018 used this promoter to control the release of the antimicrobials lysostaphin and pyocin.
-The HlyA tag is a secretion signal peptide which is used for secretion via the Type 1 secretion pathway of gram-negative bacteria.
+[[File:BBa_K2812002_SBD_Assay.jpg.png|125px|thumb|right|Figure 1: SDS-PAGE gel of pBAD-ara promotor-Lysostaphin-thrombin-HlyA-His-tag construct protein expression experiment.]]
+===Protein Expression===
+Protein expression experiments were performed to characterize the functionality of the pBAD-ara promotor. To allow expression of the construct under the control of arabinose, it was assembled together with the <partinfo>BBa_K2812004</partinfo> biobrick via Gibson assembly. After successful transformation into ''E. coli'' BL21 (DE3), the bacteria were cultured in LB medium at 37 °C. Prior to induction, a sample was taken (0) to establish the gene expression pattern of uninduced bacteria. One sample was induced at an OD600 of 0.5-0.8 by adding 2 mM arabinose to induce expression of the recombinant protein, also at 37 °C. Another sample was not induced as control. Samples were taken 4 hours after induction of both an induced (4+) and an uninduced culture (4-). SDS samples were prepared and loaded onto a polyacrylamide gel to yield the SDS-PAGE results that can be seen in figure 1.
+====Conclusion====
+The biobrick <partinfo>BBa_K2812004</partinfo> should in theory result in the overexpression of a protein with a length of 50 kDa. However, as can be read in the extensive characterisation of <partinfo>BBa_K2812004</partinfo>, self-cleavage of the fusion protein is observed, generating a part with a mass of 23 kDa and a part with a mass of 27 kDa. In the induced sample, two bands slightly below and above 25 kDa can be observed, which are not present at all in the uninduced sample. This confirms the succesful induction of protein expression of the <partinfo>BBa_K2812004</partinfo> construct, demonstrating that our pBAD-ara promotor is functional.
 <!-- Add more about the biology of this part here
-===Usage and Biology===
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K2812002 SequenceAndFeatures</partinfo>