Cloning of our device for optimizing extracellular expression using PelB/Ompt secretion tags under different Anderson promoters:
We used pSBIK3.m1 and pSB1C3 for cloning of our designed constructs. We synthesised our DNA construct via IDT. We added restriction enzyme site for Ecor1 and Pst1 in our synthesised double stranded DNA. We performed restriction enzyme digestion for both our double stranded DNA and backbones with Ecor1 and Pst1. These were ligated together.
These are the sequences which we cloned but are not submitted with iGEM registry.
pBS1K3.m1 ( JA23100->pelB-LINKER-sfGFP-HIS-STOP)
pBS1K3.m1 ( JA23102->ompT-LINKER-sfGFP-HIS-STOP)
Furthermore we have analyzed using different techniques to detect if our construct facilitates extracellular expression of sfGFP.
Detecting protein secretion using different staining techniques in E.coli (DH5 alpha and Rosetta strain)
E.coli (DH5 alpha)
We first decided to test whether our cloned product could produce extracellular protein. We chose E.coli(DH5 alpha) as our first bacterial production system. We picked colonies and set Luria broth (LB) corresponding to the constructs as shown in the scheme below. We separated supernatant and pellet corresponding to each sample. These samples were further analyzed for protein content.
Ponceau S is a sodium salt of a diazo dye of a light red color, it is used a stain for rapid reversible detection of protein bands on nitrocellulose membrane(Western blotting).
We were able to detect protein bands corresponding to pellet for construct containing JA23100/JA23102/JA23105 upstream of PelB and sfGFP. In fig1.1 the well corresponding to these construct is represented by 1,2,3. Size of protein obtained was variable. Thus, no major conclusion could be made about the nature of protein at this point. Whereas we found no or not significant quantity of ponceau staining for pellet or supernatant for construct containing JA23102/JA23105 upstream of OmpT and sfGFP which are represented by 4,5,9,10 in Fig 1.1.
Following ponceau staining we performed western blotting using anti HIS (Histidine) antibody.
Detection of our secreted protein using antibody against HIS tag.
We used Horseradish peroxidase (HRP) conjugated anti HIS antibody to detect whether our protein is secreted out. We used varying strength of Anderson promoter with different secretion tags as described in previous experiment . We found that unlike the result obtained in ponceau staining we found no band corresponding to our protein of interest (~`35kDa) in pellet of the e.coli(DH5 alpha) cloned with our devices. Whereas, we found clear bands in 6,9,10 (`35kDa) well (fig 1.2). Whereas, faint bands for devices corresponding to well 7,8 were. This suggest that our device are able to drive extracellular expression of sfGFP via e.coli(DH5 alpha). J23100 was more efficient than J23102/J23105 in extracellular expression of protein via PelB tag. We found ompT tag was efficient in facilitating extracellular expression via both J23102 and J23105 promoters.
Fig.1.2 Western Blotting via Anti-HIS antibody to detect secretion of a protein via different secretion tags pelB/ompT under varying strengths of promoters.
One of the reason for low extracellular expression via pelB secretion tags might be due to protease activity in the periplasm in e.coli(DH5 alpha) . In order to optimize extracellular expression via PelB secretion tags we also tried to check extracellular expression effieciency of our devices in E.coli Rosetta(DE3) strain. This strain is known for it’s high expression efficiency. As it lacked ompT receptor we would not be able to obtain extracellular expression via devices consisting of ompT secretion tags using this strain.
E.coli Rosetta (DE3)
We transformed the following constructs into Rosetta(DE3) strain of e.coli. We observed that there was a significant improvement in number of colonies (200>) for the transformed devices in comparison to transformation of same set of devices in e.coli(DH5 alpha). (Fig 2.1)
Fig.2.1 Transformation of devices in Rosetta(DE3). We found significant increase in the number of colonies as compared to transformation done in E.Coli DH5α for the devices facilitating secretion of sfGFP via pelB tag.
We picked colonies from the previous batch of transformation and performed coomassie staining to see if we could obtain any band to our expected size for exported protein (~35kDa).
We tried to see presence of protein both in pellet and supernatant. We observed a band corresponding to the size of our protein in samples prepared from pellet corresponding to each device(Fig 2.2). But we could not find a very significant intensity of band in samples prepared from supernatant corresponding to each device’s culture.
We will be performing western blotting using anti HIS antibody for these set of devices to definitively report about the efficiency of protein secretion via pelB secretion tag under JA23100/J23102/J23105 promoters.
Fig. 2.2 Coomasie staining for pelB secretion tag mediated extracellular secretion of protein.
Characterizing extracellular secretion of super folded green fluorescent protein(sfGFP)
To quantify the extracellular secretion of our protein we also measured fluorescence reading and OD reading of our constructs at different time points. Below is the layout of our experiment.
We have characterized devices which facilitate extracellular expression via pelB tag under J23100/J23102/J23105 promoter.
Effects of our device on growth (OD at 600nm) and the corresponding fluorescence reading (485 nm excitation and 530 nm emission wavelength) at different time points:
We tried to observe growth and fluorescence of e.coli(DH5 alpha) when cloned with our devices at five different time points. At every time point we made two separate sample: 1) Whole culture 2) Supernatant obtained after centrifugation of the same sample at that time point. We obtained OD at 600nm and fluorescence reading for two colonies(three replicate per colony).
Though the growth kept increasing till twenty two hours (Fig2.2), corresponding reading for fluorescence in supernatant and whole culture did not vary in any defined pattern. The range of variation of fluorescence was also narrow. Low intensity of fluorescence even at high OD(600nm) might be due to multiple reasons, one of them could be longer hours of incubation might lead to more cell death and release of protease and even addition of Protease inhibitor might not suffice for degradation of protein. Thus, there needs to be additional condition optimized for stabilizing protein half life in supernatant to better characterize it’s fluorescence or it’s working.
A) OD at 600nm
B) Fluoroscent at excitation wavelength 485nm and emission wavelength 530nm for culture.
C) Fluoroscent at excitation wavelength 485nm and emission wavelength 530nm for supernatant.