iGEM 2018 — Team IIT_Kanpur

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# Team:IIT Kanpur/Notebook

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**Designed construct for secretion assay :
Through July**

**August 28, 2018**

1. Transformation of all J. Anderson
   promoter
2. RE Digestion of received
   constructs with XbaI+SpeI for cloning purpose.

**August 30, 2018**

1. Mini prep of J23100, J23114,
   J23102, J23110 (J. Anderson’s promoter)

**August 31, 2018**

1. RE Digestion (With XbaI+SpeI)  of

pelB
sfGFP

pelB
linker sfGFP

ompT
sfGFP

ompT
linker sfGFP

      2.  RE Digestion of Kanamycin backbone
with with XbaI+SpeI

      3. Transformation of JA promoter.

**September 2, 2018**

1. RE Digestion of Plasmid Backbone
2. Ligation of blunt ended Kanamycin
   backbone of received constructs.

**September 4, 2018**

1. Transformation of J23105

**September 7, 2018**

1. Phosphorylation of 5’ ends of

            pelB-linker-sfGFP

            ompT-linker-sfGFP

**September 8, 2018**

1. Ligation of phosphorylated
   constructs with Kanamycin backbone.

**September 9, 2018**

1. Transformation of ligation
   performed previous day
2. Culture of JA23105, JA23102,
   JA23114, JA23110

**September 10, 2018**

1. Mini prep of JA23105, JA23102,
   JA23114, JA23110

**September 12, 2018**

1. Phosphorylation of
   pelB-linker-sfGFP and ompT-linker-sfGFP (with changed buffer and new
   enzymes)

**September 13, 2018**

1. Ligation of phosphorylated
   constructs with Kanamycin backbone

**September 18, 2018**

1. Mini prep of ompT-linker colonies.
   (Picked from previous ligation)

**September 20, 2018**

1. RE Digestion of ompT-linker with
   SpeI to check the enzyme.

**September 21, 2018**

1. Transformation of SDSa backbone
   from plasmids gifted by team TecCem
2. RE digestion repeated (as done on
   previous day, with new enzymes)

**September 25, 2018**

1. RE digestion of JA100 pelB linker
   sfGFP with EcorI and PstI.

**September 26, 2018**

1. Ligation reaction of digested
   construct in Chloramphenicol resistance containing backbone

**September 27, 2018**

1.    Ligation reaction repeated.

**September 28, 2018**

1.    Ligation reaction with changed parameters for the same insert and
vector as on previous day.

**September 29, 2018**

1. Transformation of both ligations
   done on previous day
2. Transformation of plasmid
   containing SDSa and plasmid containing JA102 promoter
3. RE digestion of received construct
   with EcorI and PstI

**October 1, 2018**

1. RE Digestion of pSBIK3.m1, pSB1A3
   and received constructs from IDT with EcorI and PstI
2. Ligation reaction of IDT
   constructs in pSBIK3.m1and pSB1A3

**October 2, 2018**

1. Transformation for the above
   ligation.
2. RE Digestion JA102 backbone with
   EcorI and ligating the linearized template to check whether ligase is
   working.

**October 4, 2018**

1. RE Digestion of IDT constructs
   with EcorI and PstI
2. RE Digestion of template pSBIK3
   and pSB1C3 with EcorI and PstI
3. Transformation of SDSa
4. Culture of SDSa colonies
5. Ligation of digested templates.

**October 6, 2018**

1. Transformation of all constructs
   in Chloramphenicol and Kanamycin backbones

**October 7, 2018**

1. Culture set for SDSa transformed
   colonies and experiment for quantitating protein secretion from our cloned
   constructs.
2. RE Digestion of backbone

**October 8, 2018**

1. Promoter sfGFP analysis of
   JA102-pelB-ompT
2. LB chloramphenicol plates were
   made
3. Transformation of JA100, JA102,
   JA105, JA108-pelB and JA102, JA105-ompT containing IDT constructs.
4. SDSa amplification using PCR
5. Culture of constructs for shipping
6. Pancau Staining for checking
   protein secretion via JA102  pelB/ompT containing backbones

**October 9, 2018**

1. PCR amplification of SDSa gene.
2. Mini prep of JA102 pelB,
   JA105-pelB/ompT containing construct
3. Interlab experiment doe
   quantitating protein secretion performed for JA102 pelB/ompT ,
4. Culture set for other quantitative
   experiments
5. Western blot for JA102 pelB/ompT

**October 10, 2018**

1. All parts were shipped.
2. Anti GFP staining for
   JA102/JA105/JA100 pelB, JA102/105 ompT
3. Fluorescence and OD measurement at
   different time points for above constructs.
4. Experiment for effects of SDS on
   growth of E.coli(DH5 alpha)

**October 11, 2018**

1. Fluorescence and OD measurement at
   different time points for above constructs.
2. Western Blot repeated with
   supernatant only.

October 13, 2018

1. Silver staining of
   JA102/JA105/JA100 pelB, JA102/105 ompT
2. Culture set for western blot via
   anti HIS antibody.

**October 15, 2018**

1. Western Blot staining with HIS
   antibody.
2. 50ml culture of JA105 pelB
   containing secretion assay construct.

**October 16, 2018**

1. Transformation of all cloned
   construct in Rosetta strain or E.coli (BL21)
2. Western blot using anti HIS
   antibody

**October 17, 2018**

1. Set culture for colonies obtained.

**October 18, 2018**

1. Ponceau staining of the
   constructs.
2. Western blot using anti HIS
   antibody.

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# Team:IIT Kanpur/Experiments

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  [References](https://2018.igem.org/Team%3AIIT_Kanpur/References)
* Modelling

  [Model Overview](https://2018.igem.org/Team%3AIIT_Kanpur/Model)
* Outreach

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* Parts and
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  [Notebook](https://2018.igem.org/Team%3AIIT_Kanpur/Notebook)
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  [InterLab](https://2018.igem.org/Team%3AIIT_Kanpur/InterLab)
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# Team:IIT Kanpur/Results

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  [References](https://2018.igem.org/Team%3AIIT_Kanpur/References)
* Modelling

  [Model Overview](https://2018.igem.org/Team%3AIIT_Kanpur/Model)
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# Results

### 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 biobricks we submitted in iGEM registry.
[BBa\_K2874000](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2874000)
[BBa\_K2874001](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2874001)
[BBa\_K2874002](http://parts.igem.org/wiki/index.php?title=Part:BBa_K2874002)

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.

![](https://static.igem.org/mediawiki/2018/c/c2/T--IIT_Kanpur--Results02.png)

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).

![](https://static.igem.org/mediawiki/2018/0/0d/T--IIT_Kanpur--Results04.jpg)

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.

![](
https://static.igem.org/mediawiki/2018/5/59/T--IIT_Kanpur--Results05.tif)

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) (In Progress)

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)

![](https://static.igem.org/mediawiki/2018/c/cb/T--IIT_Kanpur--Results31.jpg)
![](https://static.igem.org/mediawiki/2018/7/7f/T--IIT_Kanpur--Results32.jpg)
![](https://static.igem.org/mediawiki/2018/3/39/T--IIT_Kanpur--Results33.jpg)

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.

![](https://static.igem.org/mediawiki/2018/5/5d/T--IIT_Kanpur--Rosetta2.png)

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.

![](https://static.igem.org/mediawiki/2018/7/71/T--IIT_Kanpur--Results01.png)

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.

![](https://static.igem.org/mediawiki/2018/d/d9/T--IIT_Kanpur--Results22.png)
![](https://static.igem.org/mediawiki/2018/4/48/T--IIT_Kanpur--Results23.png)
![](https://static.igem.org/mediawiki/2018/d/d0/T--IIT_Kanpur--Results25.png)

Fig.2.2
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.

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