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Part:BBa_K3088001:Experience

Designed by: Syailendra Karuna Sugito, Elvan Wiyarta, Eko Ngadiono, Refael Alfa Budiman, Aditya Parawangsa   Group: iGEM19_UI_Indonesia   (2019-10-07)
Revision as of 17:59, 21 October 2019 by Enga2 (Talk | contribs) (→‎Applications of BBa_K3088001)


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Applications of BBa_K3088001

Expression of HB-EGF/EnvZ (HE) + OmpC/GFP (OG)

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 and Normal LB 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.

We also perform MagneHis on HE to further confirm the presence of HE containing HisTag (Figure 1). The resulted MagneHis band was really thin confirming HE is indeed expressed in Low Salt Medium and TB medium. Unfortunately, MagneHis result on LB medium 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.


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 (Figure 2). The reason the GFP protein can not be observed may be due to short duration of soaking in Page Blue.

Figure 2. The pelleted TOP 10 bacteria transformed with HE-OG plasmid expressing GFP where C1 means Colony 1 and C2 means Colony 2.


Characterising HBEGF/EnvZ - OmpC/GFP binding potential with Heparin

Our experiment showed the highest MEFL/ particle was observed in TOP10 which was induced with heparin diluted in 1:10000. The MEFL/particle decreases as dilution factor is reduced to 1:1000 and 1:100, respectively. By comparing the MEFL/particle of the heparin-induced-group with the non-heparin-induced-group (control), we can conclude that the induction of heparin significantly increases the number of fluorescence (GFP) expression. This proves that that upon binding of heparin sodium to our chimeric protein (HB-EGF/EnvZ), more OmpR is phosphorylated that leads to increased activation of OmpC promoter and the GFP gene downstream the OmpC promoter is successfully expressed. Our chimeric protein works !

Fluorescence reading of our blank shows a significant amount of fluorescence which might be due to the background fluorescence of the medium emits similar wavelength with GFP. (2)

Initially, our hypothesis was the more heparin is present, the expression of GFP will increase in similar manner. However, the expression of GFP is increased when the dilution factor is increased from 1/100, 1/1000, and 1/10000. The increase is quite significant between 1/1000 and 1/10000. However, we suspect that decrease of GFP expression as the dilution factor is decrease may be caused by antimicrobial activity of heparin.(3,4) According to Rosett W3, the heparin concentration needed to inhibit bacterial growth of Escherichia coli is ≤ 500 U/ml. Our 1/100 concentration of heparin sodium 5000 U/ml is 50 U/ml. The antimicrobial activity of heparin might inhibit the growth and expression of GFP and the concentration of heparin needed to induce the maximum expression of GFP is ≤ 0.5 U/ml (1/10.000 dilution).


References:

1. 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 2. Electronic Y. Spreading generally increases with lower cell ­ cell adhesion and higher cell ­ substrate adhesion . However , strong substrate adhesion can adversely affect cell movement , and modeling suggests an optimum substrate adhesiveness for spreading ( Xu et al. Phys Biol. 2016;12(2010):11–2. 3. Weijmer MC. Superior antimicrobial activity of trisodium citrate over heparin for catheter locking. Nephrol Dial Transplant. 2002 Dec 1;17(12):2189–95. 4. Rosett W, Hodges GR. Antimicrobial activity of heparin. J Clin Microbiol [Internet]. 1980 Jan [cited 2019 Oct 21];11(1):30–4. Available from: http://www.ncbi.nlm.nih.gov/pubmed/6766462

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