Difference between revisions of "Part:BBa K3088001"

Latest revision as of 15:25, 21 October 2019

HBEGF/ENVZ Chimeric Complete

Heparin-binding EGF-like growth factor (HB-EGF) is a unique receptor of the diphtheria toxin. EnvZ, on the other hand, is a member of two-component regulatory system that is important for bacteria osmoregulation. When diphteria toxin binds to HB-EGF, it will phosphorylate EnvZ and activates the signalling cascade pathway. Once EnvZ is phosphorylated, OmpR (a transcription factor of OmpC promoter) will also be phosphorylated. The phosphorylated OmpR will initiate OmpC transcription and express the gene following OmpC promoter. This is the complete part containing the appropriate promoter T5 lac promoter (BBa_K592008), Ribosomal Binding Site (BBa_B0034), our HBEGF/EnvZ chimeric protein (BBa_K3088001), Histidine tag (BBa_K1223006), and double terminator (BBa_B0010 and BBa_B0012)

Usage and Biology

This part when combined with OMPC/GFP part (BBa_K769001) can be used for diphteria toxin detection with observable green fluorescent protein.

Consideration of using TOP10 bacteria in LB medium for characterizing HBEGF/EnvZ - OmpC/GFP

There are several considerations why we use TOP10 bacteria in LB medium for HE-OG characterization with heparin. Firstly, our modelling was designed based on K12 bacteria which includes TOP10. We did not include DH5a because the results of the OMPC-GFP (BBa_K769001) expression in DH5a showed inconsistencies when the sodium concentration is increased. TB medium and low salt medium was also omitted to exclude any conditions which may affect the basal metabolism of the bacteria because these mediums are modified medium less suitable for optimal bacteria environment. Therefore, in order to measure the HE-OG expression for the very first time, we would like to use optimal environment (LB medium) as well as TOP10 bacteria to maximize the result.

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]

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 This part when combined with OMPC/GFP part (BBa_K769001) can be used for diphteria toxin detection with observable green fluorescent protein.
-<b>Expression of HB-EGF/EnvZ (HE) + OmpC/GFP (OG)</b>
-Before we measure the fluorescence of the system, we would like to check the existence of the proteins (HE & OG). We performed SDS-PAGE to measure the reconfirm the size of HE and OG. In Figure 1, we can observe 2 bands that are missed in the wildtype (WT) bacteria. The bands have the size of +/- 90kDA and 43 kDA, respectively. The size of this band is consistent with the size of HE (42.332 kDA) that we already predict by it’s structural modelling. To presence of 90 kDA band also confirms that HE presents in its dimeric form and attached to the membrane. From the thickness of the band, it is shown that TB broth has the thickest band compared to Low Salt LB Medium where Normal LB Medium has the thinnest band among other medium. In the TB & LB medium, the thickness of the HE band gradually increase from row 1 to row 4, consistent with the duration after IPTG is added. In Figure 5, we can also observe the presence of 43 kDA band. This would confirm that this band is a monomer of our chimeric protein. Based on literature, we know that not all EnvZ will form dimer because of the existence of the dimer-monomer equilibrium.(1) From this we could conclude that each of the 90 kDA band and 43 kDA is our chimeric protein on dimer and monomer structure respectively.
+<b>Consideration of using TOP10 bacteria in LB medium for characterizing HBEGF/EnvZ - OmpC/GFP </b>
-We also perform MagneHis on HE to further confirm the presence of HE containing HisTag Figure 5. The resulted MagneHis band was really thin confirming HE is indeed expressed in Low Salt Medium and TB medium. Unfortunately, MagneHis result on DH5a shows several bands which may be due to un-specific binding of the MagneHis to HE. Another explanation may be due to saturated concentration of bacteria and thus during the washing part, the washing is not completely clean. Therefore, during the elution, some of the leftover bacterial protein get eluted. It can be observed that HE is still present.
+There are several considerations why we use TOP10 bacteria in LB medium for HE-OG characterization with heparin. Firstly, our modelling was designed based on K12 bacteria which includes TOP10. We did not include DH5a because the results of the OMPC-GFP (BBa_K769001) expression in DH5a showed inconsistencies when the sodium concentration is increased. TB medium and low salt medium was also omitted to exclude any conditions which may affect the basal metabolism of the bacteria because these mediums are modified medium less suitable for optimal bacteria environment. Therefore, in order to measure the HE-OG expression for the very first time, we would like to use optimal environment (LB medium) as well as TOP10 bacteria to maximize the result.
-[[File:HEOG SDS.jpg|700px|thumb|centre|Figure 1. This shows the SDS-PAGE of TOP10 bacteria grown in TB medium, LB medium and Low Salt LB medium. The lane ‘-’ indicates TOP10 before induced by IPTG. The lane ‘1-4’ indicates sample collected at hour 1 to 4 after IPTG has been added. The lane A, B and C indicates MagneHis of TOP10 in TB medium, LB medium, and Low Salt LB medium, respectively. The WT indicates TOP10 wildtype.
-]]
-Unfortunately in this SDS-PAGE, the GFP protein can not be seen. However, the presence of GFP can be confirmed when the bacteria is pelleted and observed under UV light. The reason the GFP protein can not be observed may be due to short duration of soaking in Page Blue.
-References:
-. Khorchid A, Inouye M, Ikura M. Structural characterization of Escherichia coli sensor histidine kinase EnvZ: the periplasmic C-terminal core domain is critical for homodimerization. Biochem J [Internet]. 2005 Jan 1 [cited 2019 Oct 20];385(Pt 1):255–64. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15357641
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