iGEM 2022 — Team iit-delhi

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# Notebook

# Week 1

We started work in the wet lab in the first week of April. With a lot of us starting off in the lab for the first time, independent lab work was a daunting task in itself. However, under the supervision of our Advisor, we dedicated the first couple of
weeks to learning lab etiquette and safety and security protocols. In the first week, we prepared the cloning strategy while simultaneously learning all the precautions, safety guidelines, lab etiquette, and equipment usage.

# Week 2

During this week, we designed the primers and ordered them following an intensive literature review. We also ordered the plasmids required- pEPR1, pSR2, pPS17, and pRSG43. We organized the reagents and streaked plates. For this, we prepared LA and poured
it on plates. We used LA for streaking of  *E. coli*  DH5ɑ cells, LA+Kan50 for pEPR1, pSR2, and pRSG43, and LA+spec40 for pPS17. Further, we inoculated tubes of pEPR1 in LB. We then kept the tubes in an incubator at 37ºC at 180 rpm.

# Week 3

From the tubes inoculated by us, we performed plasmid isolation of pEPR1 to obtain a stock of isolated plasmids for use in the biosensor. We checked for the proper isolation of the plasmids by performing gel runs on 1% agarose gel with EtBr as the staining
dye. We also checked the concentrations of the isolated plasmids on the nanodrop. The plasmid was stored at 0ºC for use later.

# Week 4

We continued the isolation of plasmid pEPR1. Further, we also received the ordered primers for PbrR- P1 to P12. We started with the overlap extension PCRs of the primers. We set up OE PCRs of P1-P2, P3-P4, P5-P6, P7-P8, P9-P10, and P11-P12. We confirmed
the correct overlap by running the samples on 2% agarose gel and comparing the sizes.

# Week 5

Overlap extensions from templates derived in the previous week were set up. The reactions were now set for P1-P4, P5-P8, and P9-P12. The results were checked on 2% agarose gel. Simultaneously, another batch of step 1 OE PCRs were set up again to keep
a stock ready.

# Week 6

The final OE PCR was set up for P1-P12, however, upon gel run, the size did not match with the required size. Another PCR was set from the remaining stock left from the previous templates. Simultaneously, the step 2 OE PCR was set up for the second batch.
The final step was also set up for the second batch.

# Week 7

The results were now visualized on 12% PAGE for better resolution and comparison of sizes. pEPR1 was set up for digestion using enzymes NsiI and XbaI. Simultaneously, competent cell solutions were prepared. The digestion was checked by running the sample
on 1.5% agarose gel and looking for a linearized band. Since the restriction sites of NsiI and XbaI were too close, the release could not be seen visually on the gel.  *E. coli*  DH5ɑ was inoculated in 2% 5mL LB and left in the incubator at 37ºC
at 180 rpm on Saturday.

# Week 8

Batches of competent cells were prepared every alternate day, and their transformation efficiency was checked. The first batch of competent cells was prepared on the third try after 2 failed attempts. These were then stored at -80ºC. Simultaneously, OE
PCR reactions were continued. Due to the inability to see a release, sequential digestion was performed for the vector with enzymes NsiI and XbaI. The digestion was finally successful on the fourth attempt. Insert digestion was set up using
enzymes NsiI and XbaI.

# Week 9

OE PCRs were set up continuously, and competent cell preparation, and vector digestion were also done every day to prepare stock. First cycle of ligation-transformation was set up using the prepared vector and insert digest. No colonies were observed
on plating. Thus, the experiment cycle was repeated due to lack of results.

# Week 10

We started preparing our plasmid pEPR1, starting its isolation followed by its digestion. The DH5a competent cells were prepared and transformation efficiency was checked which yielded an empty plate. The OE PCRs results were observed on 12% PAGE gels.
The fragments F5-F6, F7-F8, and F9-F10 were successful as seen in the picture. PAGE gel was then washed with RO water wrapped in a paper towel and stored in the freezer at 4ºC.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/dna-page.png)

# Week 11

PCR was set for PbrR promoter,but as we didn't know the optimal annealing temperature we set it for three different annealing temperature-60,61,62.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/results-gel-run.png)

The insert was digested by different methods-
i) Double digesting it with NsiI and XbaI
ii) Double-step single digestion.
This was done to optimize the digestion protocol and to check if both the enzyme were properly digesting
the plasmid in 2hrs, which was found to be true.

Transformation efficiency was performed 3 times, and we found colonies 2 times. We used only 1 plate as the colonies in the other plate were very few.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/week11-2.png)
![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/week11-3.png)

# Week 12

This week we performed ligation and transformation three times, observing no growth on the plate. PbrR promotor was prepared as an insert and amplified in PCR. Competent cells were prepared again to create a stock of comp cells.

# Week 13

The fragments F5-F6, F7-F8, and F9-F10 were again set for OE PCR, we shifted to Phusion buffer to get better results. Vector digestion was set up and was successful. The PCR was found to be unsuccessful and the OE PCR was set again.

# Week 14

For insert preparation, we then shifted to blue white screening of PGMT colonies and tried the vector preparation

# Week 15

Due to failed results of OE PCR we decided to speed up things and ordered the PbrR gene and the MBD (metal binding domain) of the PbrR gene. This week we performed the amplification of these using PCR from their stock solutions.

Also simultaneously the pSR2 was digested and Ligation and Transformation was performed yielding no results.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/week15.png)

# Week 16

We tried Ligation and Transformation again with fresh vectors and Inserts but weren’t able to find any colonies. And we were also facing issues with Insert preparation as the concentrations of our inserts are coming out very weak. So to check whether
there are any mistakes while preparing the insert, we performed insert prep twice and still failed to get good concentrations of PbrR and Metal Binding of PbrR genes.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/dna-page.png)

# Week 17

We discussed the issue with everyone and concluded that our primers might not be working properly and so prepared new working solutions of primers for both PbrR and the Metal Binding Domain of PbrR. We finally got well-concentrated inserts with the new
primer solutions. So good concentrations of inserts are prepared.

![](https://static.igem.wiki/teams/4428/wiki/awards/week-17.png)

# Week 18

We discussed with our faculty about the weak concentrations of vectors and made some minor changes in the procedure of vector preparation. Finally, we got good concentrations of vector and inserts, performed Ligation, and Transformed them into  *E.coli*
Dh5α.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/week18.png)

# Week 19

Finally, Colonies were formed. To confirm whether the clone is formed or not, we did Colony PCR and Restriction digestion of the plasmid isolated from the colonies but were unable to confirm the clone.

# Week 20

We performed Ligation and Transformation twice and found colonies on both PbrR and Metal Binding Domain of PbrR.

# Week 21

We did Colony PCR and Restriction Digestion of the plasmid isolated from the colonies and successfully confirmed the clone. And stored the colonies.

# Week 22

Now that the clone was ready, we transformed this recombinant plasmid into  *E.coli*  plysS.

![](https://static.igem.wiki/teams/4428/wiki/wet-lab/notebook/week22.png)

# Week 23

Now that the clone was ready, we transformed this recombinant plasmid into E.coli plysS.

# Week 24

Now that the clone has been confirmed and transformed into E.coli plysS, for protein expression we induced the culture of E.coli plysS (transformed with recombinant pSR2) with IPTG and collected samples for SDS PAGE. And also parallely for checking the lead adsorption capacity, before IPTG induction, we added Pb(NO3)2 to the culture and collected samples at different time intervals. Then performed lead adsorption analysis using ICP-MS.

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# Engineering

### Design

As we identified the need to address lead contamination in aqueous media, we devised a synthetic biology solution to it. We began by narrowing down to the crux of the problem by analysing exactly where current methods of recovery, implemented by lead-based
industries to treat their wastewater, lack. We studied the current strategies for lead removal, recovery and recycling. While current physicochemical methods were extremely useful at high lead concentrations, they were uneconomical and inefficient
at lower concentrations commonly found in industrially treated wastewater. These methods can bring down the lead concentration of industrial wastewater from 200-500 mg/L to 0.4 mg/L. However, even these lower concentrations of lead are extremely
harmful to life forms.

Biosorbants which not only remove lead from water but also allow for successive desorption for the recovery of metal were an ideal solution to our problem. Not only does this solution limit exposure to the heavy metal by removal from aqueous media, it
also provides the opportunity for value generation for our stakeholders i.e wastewater treatment plants which can redirect the recovered lead to industries. We began reviewing the literature for strategies to implement the cell-surface display
of desired proteins. We studied the benefits and limitations of multiple anchor proteins that help express desired proteins on the outer membrane of the cell. Finally, we identified an anchor, BclB, suitable for our system.

Additionally, we began searching for lead-binding proteins which can potentially be expressed on the cell surface to recover the lead. We identified a popular metalloregulatory protein found in C. metallidurans proteins, PbrR, as a candidate for our system.
We primarily wanted to address the need for high display efficiency on the cell surface, high binding affinity to lead and specificity for recovery. For improving the display efficiency and to reduce metabolic burden on the cell, we decided to
use only the metal binding domain (MBD) of PbrR as the cell surface protein. Since this was a much smaller region of the protein, it was hypothesised to be expressed more efficiently without metabolically burdening the cell. Additionally, in the
limited surface area of the cell membrane, a smaller protein was hypothesised to improve the display efficiency i.e the number of protein molecules displayed on the cell surface would increase providing an opportunity for higher lead adsorption.
Further, we identified another lead-binding protein called PbrR691. This is a recombinant version of PbrR known to be 1000 times more specific to Pb than to other heavy metals. We also decided to further use the metal binding domain of PbrR691
which shall improve display efficiency as well as specificity. Thus, we identified these four proteins - PbrR, PbrR MBD, PbrR691 and PbrR691 MBD - for a comparative study to develop a successful lead recovery system.

### In silico analysis

Before beginning our wet lab, we structurally modelled all of the shortlisted proteins in our dry lab to find the best protein, its metal binding region, and the lead-protein complex stability. We first generated the protein structures using their amino
acid sequences and also used confirmed structures found in the literature. We simulated these in an environment of water to closely represent our media and test the binding of lead with the protein in the environment we intend to use it. We confirmed
the fact that all these proteins fold correctly in the system. We ensured that the metal binding domain also folds correctly by itself in the absence of the rest of the protein. We further dock these proteins with lead to test their binding efficiency
and also if the binding is reversible for our purposes of desorbing the metal from the surface protein for further use. The detailed results of our in silico analysis can be found in the [Modelling section](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D).

## Build

We cloned all of our shortlisted proteins in our designed recovery plasmid. The plasmid has a pSR2 vector backbone with BclB upstream to the cloning sites of the lead-binding proteins. For cloning of the genes of Interest, we prepared inserts by digesting
the PCR amplified genes with restriction enzymes SacI and HindIII. For the vector, we digested the recovery plasmid, which already consists of our anchor, with the same enzymes. Once the vector and inserts were ready, we ligated them and transformed
the recombinant pSR2 into  *E.coli*  DH5α. We then performed the clone confirmation through colony PCR and double digestion. The results reflected that we were successfully able to clone PbrR and its MBD into the vector. Hence, we moved forward with
testing of the two clones while we continued to attempt the cloning of PbrR691 as well.

## Test

We thus commenced the testing phase of our project. We transformed our confirmed clones in the  *E. coli*  strain pLysS meant for protein expression. We tested our parameter for degree of cell surface display efficiency using SDS-PAGE. We induced the expression
of the surface proteins and then performed electrophoresis to test whether the protein was successfully being expressed. The intensity of the bands was also used to deduce the degree of expression.

To test the efficiency of lead adsorption, we have specifically designed experiments to measure lead concentrations when lead is put in a culture medium with  *E. coli.*  We used ICP-MS (inductively coupled plasma-mass-spectrometry) to measure the exact concentrations
of the element down to parts per billion. We grew  *E. coli*  until a defined concentration of cells and introduced a known concentration of lead to it. We then collected samples at multiple time points, filtered the bacterial cells by centrifugation,
and then measured the lead concentration in the supernatant. We tested multiple variables in the experiment by changing either the  *E. coli*  concentration, the initial lead concentration and having different durations of lead exposure. The details
and results of this testing process can be found [here](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D).

## Learn

The SDS-PAGE results of our engineered cells confirm the expression of the cell-surface protein PbrR. The gel for PbrR MBD doesn’t explicitly show overexpression of the domain, although we attribute it to the extremely small size of the protein as the cells containing the metal binding domain show the ability to adsorb lead in subsequent experiments. The protein expression analysis needs to be optimised for PbrR MBD to accurately detect its expression.

Our engineered bacteria were then tested on artificially Pb-contaminated deionised water demonstrating extremely promising adsorption of lead. We begin with a concentration of 400ppb of lead in the samples. This concentration is equivalent to 0.4mg/l,
the threshold concentration at which industrial recovery methods become ineffective. The treated samples, measured for lead concentration using ICP-MS, are left with almost a 40-fold reduction of the lead concentration.

While an in-depth analysis of the results can be found on the [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page, we do learn that the bacterial cells are successfully able to remove lead from the environment. Interestingly, even the uninduced cells were able to remove lead from
the environment which led us to look into the leaky expression of the protein. Thus, we dove back into the literature to see whether the same strain leads to leaky expression of the surface display protein downstream to the BclB anchor under the
T7 promoter. Indeed, S. Rangra et al. study using the same strain and construct with a different surface display protein also noted leaky expression even in the uninduced state. This helped deduce that at this concentration, even low expression
of the protein is successfully able to treat the samples. Thus, the cells could potentially show much greater adsorptive capacity at higher concentrations. This, however, raises the concern about the ability of  *E. coli*  to sustain higher lead concentrations.
For the system to be viably used at even higher concentrations of lead, this must be investigated. We also observe that a longer duration of induction of the surface protein led to poorer removal of lead from the sample. Cell death could be attributed
to this result due to cells entering their stationary phase rather than staying in the exponential growth phase, leading to poorer adsorption of lead. We also learn that the metal binding domain of PbrR works almost as well as PbrR in terms of
removing lead from the sample.

## Design

We have found that our engineered bacteria work well at concentrations of lead around 400 ppb. We also learnt that the adsorptive capacity of the engineered cells could potentially be much higher than that observed at these concentrations. These preliminary
learnings suggest that we must study the adsorptive capacity of our cells with varying lead concentrations. Simultaneously, the ability of  *E. coli*  to sustain higher lead concentrations needs to be tested to characterise if this recovery system
would be viable at these higher concentrations of lead. We would also need to investigate if this ability of cell-surface adsorption of lead allows  *E. coli*  to sustain higher lead concentrations than the wild type. Additionally, we are yet to test
multiple other parameters like varying the concentration of engineered cells to treat the samples, varying the duration of induction of the cells, varying the duration of exposure to lead to identify how long this procedure of adsorption takes
and to verify if long durations of exposure to the heavy metal can be toxic to the bacteria and if we can combat it. We hoped to study the role of each of these parameters to comprehend our results better and to recognise the potential of this
system, but could not execute these experiments in the limited time frame. We hope to execute the analysis of these parameters in our next design cycle. Additionally, we have observed these results in artificially contaminated water, however,
in reality, wastewater samples would have various other interfering impurities. We need to understand how those impurities, especially other heavy metals, affect our system’s capacity to adsorb lead. Also, to shed light on our objective to find
the most efficient lead-binding protein for this recovery system, we need to observe how the two constructs fare when varying the pre-stated parameters to conclusively identify the best candidate.

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# Protocols

### List of Protocols

1. [LB (nutrient media) preparation](#lb_prep)
2. [LA (solid media) preparation](#la_prep)
3. [Plasmid isolation (from  *E. coli* )](#pi)
   [* Plasmid isolation solution I preparation](#pi_soln1)
   [* Plasmid isolation solution II preparation](#pi_soln2)
   [* Plasmid isolation solution III preparation](#pi_soln3)
4. [Restriction Enzyme Digestion](#re_digest)
5. [Agarose Gel Electrophoresis](#age)
6. [*E. coli* competent cells preparation](#ccp)
   * [CC solution I preparation](#cc_soln1)
   * [CC solution II preparation](#cc_soln2)
   * [CC solution III preparation](#cc_soln3)
7. [Transformation efficiency for competent cells](#transfromation)
8. [Ligation and Transformation](#lig_trans)
9. [SDS-PAGE](#sds_page)
10. [PCR](#pcr)
11. [IPTG Induction](#iptg)
12. [Lead Adsoprtion Analysis](#lead_absop)
13. [Primer Revival](#primer)
14. [Gene Revival](#gene_revival)

### LB (nutrient media) preparation

1. Add 2 g of Luria Broth mixture to 100 mL of distilled water to make 2% w/v solution in an Erlenmeyer flask.

2. Mix well to form a uniform solution.
3. Make an air-tight cotton plug and close the mouth of the flask using the plug.
4. Autoclave the flask at 121℃, 15 psi pressure for 20-25 minutes. (Cover the cotton plug and the mouth of the
flask with a piece of Aluminum foil before keeping inside the autoclave).
5. Sterilized liquid media is ready for use.

### LA (solid media) preparation

1. Add 2 g of Luria Broth mixture to 100 mL of distilled water to make 2% w/v solution in an Erlenmeyer flask.

2. Mix well to form a uniform solution.
3. Add 1.5 g of Agar Agar powder to the solution and mix well to make a uniform 1.5% w/v solution.
4. Make an air-tight cotton plug and close the mouth of the flask using the plug.
5. Autoclave the flask at 121℃, 15 psi pressure for 20-25 minutes. (Cover the cotton plug and the mouth of the
flask with a piece of Aluminum foil before keeping inside the autoclave).
6. While the media is still in liquid form, pour ~25 mL of media in 4 sterile petri plates each inside a laminar
hood.
7. Let the media solidify inside the laminar hood.
8. Once solidified, cover the petri plates with their lids and seal them using parafilm.
9. Sterile solid media is ready for use.

### Plasmid isolation (from *E. coli*)

1. Inoculate a single colony of  *E. coli*  in 5 mL LB in a test tube with appropriate antibiotics.

2. Incubate overnight at 37℃ and 180 rpm.
3. Harvest the cells by centrifugation at 12000 rpm for 2 minutes (at room temperature).
4. Discard the supernatant and collect the pellet.
5. Add 1 mL milli-Q water and centrifuge at 12000 rpm for 2 minutes at room temperature.
6. Discard the supernatant and collect the pellet.
7. Resuspend the pellet in 100 μL of Solution I (ice cold).
8. Incubate for 5 minutes in ice.
9. Add freshly prepared Solution II (200 μL).
10. Mix gently by inverting the tubes 4-5 times.
11. Add 150 μL of ice cold Solution III and mix by inverting 3-4 times and keep on ice for 5 minutes.
12. Centrifuge at 12000 rpm at 4℃ for 15 minutes.
13. Transfer the supernatant to a fresh tube.
14. Add 0.7 volume isopropanol (0.7 volume of the total volume of supernatant).
15. Mix by inverting or vortexing.
16. Keep at room temperature for 10 minutes.
17. Centrifuge at 12000 rpm at 4℃ for 15 minutes.
18. Discard isopropanol (supernatant) and add 1 mL 70% ethanol to wash the pellet.
19. Centrifuge at 12000 rpm at 4℃ for 2 minutes.
20. Air dry the pellet overnight.
21. Dissolve the pellet in 40 μL of Elution buffer (10mM Tris-Cl, pH 8.5).
22. Add 0.2 μL RNase.
23. Incubate for 1 hour at 37℃.
24. Store the plasmid at 0℃.

### Plasmid Isolation solution I preparation

For 100 mL, pH 8,

1. Add the following to an empty Erlenmeyer flask:
   * 0.908 g glucose
   * 0.30283 g Tris
   * 0.40947 g EDTA
2. Add 10 mL distilled water and mix well.
3. Maintain pH at 8 using HCl.
4. Make up the volume to 100 mL.

### Plasmid Isolation solution II preparation

Important note: Use freshly prepared solution II

For 1 mL,

1. Add the following to an empty falcon:
   * 100 μL 2 N NaOH
   * 100 μL 10% SDS
   * 800 μL Milli-Q
2. Mix well.

### Plasmid Isolation solution III preparation

For 100 mL, pH 4.8,

1. Add 29.442 g Potassium acetate to an empty Erlenmeyer flask.
2. Add 10 mL distilled water and mix well.
3. Maintain pH at 4.8 using glacial acetic acid.
4. Make up the volume to 100 mL.

### Restriction Enzyme Digestion

1. 25 μL Reaction Mixture:

2. Plasmid/genome to be digested (upto 1 μg., volume decided according to concentration).
3. Suitable buffer - 2.5 μL (chosen according to enzymes)
4. Enzyme 1 - 0.5 to 1 μL (volume according to activity)
5. Enzyme 2 - 0.5 to 1 μL (volume according to activity) [added if double digestion needs to be done]
6. milli-Q water - make up volume to 25 μL

**Steps:**
1. Make the suitable reaction mixture and mix slowly by gentle pipetting.
2. Incubate the mixture at 37℃ for 1-2 hours.
3. Perform agarose gel electrophoresis of 1 kb ladder, undigested plasmid/genome and digested plasmid/genome on
1% agarose gel. (run 2-3 μL of sample at 1 hour to check if digestion is complete, and full load when digestion
is complete).
4. Cut the required band and perform gel extraction to obtain the required digest.

### Agarose Gel Electrophoresis

**Gel preparation (1% w/v):**
1. Measure 0.5 g Agarose powder on a weighing balance.

2. Add it to an Erlenmeyer flask.
3. Add 49 mL distilled water and 1 mL TAE buffer (50 volume) to the flask.
4. Mix well, and microwave the mixture for 1-1.5 minutes, till the agarose completely dissolves to form a clear
solution.
5. Let the solution cool till the flask is bearable to touch.
6. Add 2 μL EtBr carefully and mix well. (Use gloves while handling EtBr)
7. Pour the mixture in an agarose gel setting tray.
8. Place the comb to form the required number of wells on the setting tray.
9. Let the gel solidify.

**Electrophoresis:**
1. Place the solidified gel in the gel run apparatus.
2. Pour 1 volume TAE buffer in the gel run apparatus till it completely covers the surface of the gel.
3. Load the gel with the required samples (mix 1x loading dye with each of the samples before loading) and the
suitable ladder.
4. Connect the electrodes of the gel run apparatus with the power pack (wells towards the negative electrode).

5. Switch the power pack on and let the gel run till 3/4th length of the gel.
6. Observe the gel under UV transilluminator to identify the required bands.

### *E. coli* competent cells preparation:

Important note: All steps to be carried out in a sterile, cold environment

1. For primary culture, isolate a single colony of  *E. coli*  DH5ɑ in 5 mL LB. Incubate overnight at 37℃ at 180
   rpm.
2. Transfer 1% inoculum (1 mL for 100 mL media) in 100 mL LB to prepare secondary culture. Incubate at 37℃ for
   2-3 hours till OD reaches 0.4-0.6.
3. Chill on ice for 20 minutes and centrifuge at 3000 rpm for 10 minutes at 4℃ after putting in oakridge tubes.
4. Discard the supernatant and collect the pellet.
5. Resuspend the pellet in ice-cold Solution I by filling oakridge tubes to half of their volume by length.
6. Centrifuge again at 5000 rpm for 10 minutes at 4℃. Discard the supernatant and collect the pellet.
7. Add 2 mL of ice-cold Solution II.
8. Centrifuge again at 5000 rpm for 10 minutes at 4℃. Discard the supernatant and collect the pellet.
9. Resuspend the pellet in 1 mL ice-cold Solution III.
10. Aliquot 100 μL of resuspended pellet in 10 MCTs each.
11. Competent cells preparation is complete. Store at -80℃.

### CC solution I preparation

For 100 mL,

1. Add the following to 100 mL milli-Q water:
   * 1.626 g MgCl2
   * 0.294 g CaCl2
2. Mix well.
3. Store at 0℃.

### CC solution II preparation

For 50 mL,

1. Add 0.735 g CaCl2 to 50 mL milli-Q water.
2. Mix well.
3. Store at 0℃.

### CC solution III preparation

For 20 mL,

1. Add the following to 17 mL milli-Q water:
   * 3 mL Glycerol
   * 0.294 g CaCl2
2. Mix well.
3. Store at 0℃.

### Transformation Efficiency for CC

Important note: All steps to be carried out in a sterile, cold environment

1. Add 2 μL plasmid to 100 μL of competent cells.
2. Keep mixture on ice for 30 minutes.
3. Give a heat shock at 42℃ for 90 seconds.
4. Keep on ice for 10 minutes.
5. Add 1 mL LB to the mixture.
6. Incubate at 37℃ for 1.5 hours at 180 rpm.
7. Spread 100 μL of culture so obtained on an LA plate (having suitable antibiotics).
8. Incubate the plate overnight at 37℃.
9. To calculate transformation efficiency, use the following formula:
10. Transformation efficiency (cfu/μg) =
    ![](https://static.igem.wiki/teams/4428/wiki/wet-lab/cfu-formula.png)

### Ligation-Transformation

10 μL reaction mixture:
 **Test:**

1. 10x Ligase buffer - 1 μL
2. T4 DNA ligase - 1 μL
3. Vector - volume decided according to concentration (50 ng vector DNA required in reaction mixture)
4. Insert - volume decided according to concentration (insert:vector mass ratio in reaction mixture
   should be 3:1)
5. milli-Q water - make up volume to 10 μL

10 μL reaction mixture:
**Control:**

1. 10x Ligase buffer - 1 μL
2. T4 DNA ligase - 1 μL
3. Vector - volume decided according to concentration (50 ng vector DNA required in reaction mixture)
4. milli-Q water - make up volume to 10 μL

1. Make the suitable reaction mixture for both test and control. Perform the following steps for both the test
   and the control.
2. Incubate at 22℃ for 3 hours.
3. Incubate at 16℃ for 2 hours.
4. Transform the entire mix in 100 μL competent cells solution.
5. Keep the mixture on ice for 30 minutes.
6. Give a heat shock at 42℃ for 90 seconds.
7. Immediately transfer to ice for 5 minutes.
8. Add 1 mL LB.
9. Incubate at 37℃ for 1.5 hours at 180 rpm.
10. Centrifuge at 5000 rpm for 5 minutes at room temperature.
11. Remove 900-910 μL supernatant from the top so that ~100 μL supernatant is left.
12. Resuspend the pellet in the remaining 100 μL supernatant.
13. Spread the resuspended mixture on an LA plate (having suitable antibiotics).
14. Incubate the plate overnight at 37℃.

### SDS-PAGE

1. Take approximately 1 ml of bacterial culture from each of the samples and centrifuge at 12,000 rpm for 2
   min.
2. Discard the supernatant and wash the pellet with MilliQ.
3. Centrifuge the suspension and discard the supernatant.
4. Add 50μl of SDS-lysis buffer to the pellet and resuspend the pellet gently in the lysis buffer.
5. Boil the sample at 95°C for 10 min.
6. Centrifuge the suspension at 12,000 rpm for 5 min.
7. Load 15 μl of supernatant on an SDS-PAGE gel.
8. Run the gel until the dye reaches 3/4th the length of the gel at 80V.
9. Stain the gel with CBB R-250 solution for 15 min.
10. De-stain the gel with a destaining solution.

### Polymerase Chain Reaction

1. Mix the following in a single PCR tube:
   * Standard Taq buffer - 2.0 μL
   * dNTP - 1.5 μL
   * Forward Primer - 1.0 μL
   * Reverse Primer - 1.0 μL
   * Taq Polymerase - 0.2 μL
   * Template - volume decided according to concentration (1μg needed in the mixture)
   * MilliQ - make up volume to 20 μL
2. Keep the PCR vial in the thermal cycler and set the following cycle conditions:
   * Initial denaturation - 95°C - 3 mins (for standard PCR) / 10 mins (for colony PCR)
   * Denaturation - 95°C - 1 minute
   * Annealing - Annealing temperature (based on primers) - 1 minute
   * Extension - 72°C - Duration dependent on size of template (extension speed of Taq polymerase - 1
     kb/minute)
   * Final extension - 72°C - 10 minutes
   * Hold - 4°C
3. Analyze the products using gel electrophoresis.

### IPTG Induction

1. Incubate a culture of plysS + plasmid with the required insert in 5 mL of LB with suitable antibiotics,
   overnight at 37℃ at 180 rpm (primary inoculation).
2. Use 50 μL of the above culture as inoculum and inoculate another 5 mL of LB having suitable antibiotics and
   incubate at 37℃ at 180 rpm till OD reaches 0.3-0.4 (secondary inoculation).
3. Once OD reaches 0.3-0.4, collect and store (at 4℃) a 1 mL sample in a sterile MCT before inducing the
   culture with 5 μL of IPTG and further incubating it at 37℃ at 180 rpm.
4. Collect and store (at 4℃) a 1 mL sample after 3 hours, 5 hours and overnight of incubation after induction
   each.
5. For each of the collected samples, pellet down the cells by centrifugation.
6. Collect the supernatant in a separate tube and save the pellet.
7. Suspend the pellet in 80μL dye and 80μL of the supernatant in 20μL dye.
8. Heat both of them at 95℃ for 10 minutes.
9. Centrifuge both of them at 12000 rpm for 10 minutes.
10. Perform Agarose gel electrophoresis of 16-17μL of each of the two samples.

### Lead Adsorption Analysis

1. Grow overnight cultures of MBD-plysS and PbrR-plysS in 5 mL LB + Kan.
2. Use primary cultures as inoculum for secondary inoculation in 2 flasks each.
3. At 0.3 OD, induce one flask with IPTG.
4. After induction, collect 40 ml samples from both flasks at 6 hours and 12 hours after incubation.
5. Pellet down the cells from the collected samples and discard the supernatant.
6. Resuspend the cells in 40 mL Basal Salt Media.
7. To this 40mL, add 400ppb of lead nitrate solution.
8. Incubate the flasks at 37℃ at 180 rpm.
9. Collect 10 ml samples from the flasks at 3 hours, 6 hours, 9 hours and 12 hours of incubation each.
10. Centrifuge the collected samples at 5000 rpm for 10 minutes.
11. Collect 5 mL of supernatant and filter sterilize using a 0.2u filter.
12. To this 5 mL supernatant, add 5 mL of 5% conc. Nitric acid.
13. Mix the tubes gently and submit for ICP-MS.

**Minimal Media Composition:**

1. Na2HPO4 - 2 g/L
2. KH2PO4 - 1 g/L
3. NH4Cl - 3.59 g/L
4. MgCl2.7H2O - 0.4 g/L

### Primer Revival

For 20 mL,

1. Centrifuge the lyophilized primer at 12000 rpm for 10 minutes at room temperature.
2. Add elution buffer (specific volumes for specific primers) to get to a concentration of 100 μg/μL.
3. Vortex thoroughly.
4. Centrifuge at 12000 rpm for 5 minutes at room temperature.
5. To make the working solution, mix 5 μL of stock primer along with 95 μL of elution buffer.

### Gene Revival

1. Centrifuge the tube at 3000 rpm for 1 minute at room temperature to ensure that the material is at the
   bottom of the tube.
2. Add elution buffer to get to a final concentration of 10 ng/μL.
3. Vortex briefly.
4. Incubate at 50℃ for 20 minutes.
5. Briefly vortex and centrifuge.

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# Results

# Overview

We spent around 6 months in the laboratory which was the most crucial and labour-intensive part of our project. After several trials and errors, we were finally able to slowly move towards our goal. Following are the intermediate and final results that
we obtained.

## **Gene amplification and insert preparation**

For the construction of the clone, we revived the primers and genes. We amplified the genes through PCR at their respective annealing temperatures, 61°C (calculated with NEB Tm calculator) with Taq polymerase. Then they were run on a 1.8% agarose gel
alongside a 100bp ladder and extracted via gel elution. The sizes of the amplified genes were confirmed to ensure correct amplification. For the preparation of inserts, we digested the amplified genes with SacI and HindIII restriction enzymes
overnight.

![](https://static.igem.wiki/teams/4428/wiki/result/results1.png)

## **Vector Preparation**

For the preparation of the vector, we inoculated pSR2 colonies in Luria Broth containing Kanamycin (Kan) and incubated them overnight, shaking at 180rpm. Cells were harvested and pSR2 was isolated from the harvested cells. This isolated pSR2 was run on
1% agarose gel alongside a 1kb ladder. Then restriction digestion of 1.5 h was performed at 37°C with enzymes SacI and HindIII in cutsmart buffer. The Plasmid pSR2 is of size 6597 bp. After restriction digestion, a vector of length 5336 bp and
a release of 1261 bp were expected. These sizes correspond with the obtained results of gel electrophoresis of the digested plasmid confirming the vector preparation.

![](https://static.igem.wiki/teams/4428/wiki/result/results2.png)

## **Ligation and Transformation**

The vector obtained above was then ligated with the 2 prepared inserts, giving us the recombinant pSR2 which was subsequently transformed into  *E.coli*  DH5α.

![](https://static.igem.wiki/teams/4428/wiki/result/results3.png)

## **Confirmation of the clone** In order to confirm the clone, colony PCR was performed with the obtained colonies using their respective primers. When positive results for colony PCR were obtained, further confirmation was performed using restriction digestion of the recombinant plasmids. For the final confirmation, these colonies were inoculated in order to isolate the recombinant plasmid. These plasmids were further digested using SacI and HindIII enzymes to check the size of the release obtained. The size of the release was found to be the same as their respective inserts, thus confirming that the clone was successful. ![](https://static.igem.wiki/teams/4428/wiki/result/results4.png) ![](https://static.igem.wiki/teams/4428/wiki/result/results5.png)

## Testing protein expression using SDS-PAGE

The confirmed clones were then transformed in  *E. coli*  strain pLysS for proper expression of the introduced proteins. To confirm whether the proteins were successfully being expressed or not, we induced the engineered cells for different durations of time
and analysed their protein content on an SDS-PAGE gel.

![](https://static.igem.wiki/teams/4428/wiki/result/results6.png)

The SDS-PAGE results for PbrR transformed cells show the expression of the protein as we induce the sample. The uninduced sample shows no expression of the protein. The 3-hour induced sample also doesn’t show any discernable expression of the protein.
However, at 6 hours, the protein is clearly expressed and shows a significant band corresponding to 16kDa. For the overnight induced sample, the intensity of the band reduces and we attribute that to cell death in the system. However, the results
of SDS-PAGE for the PbrR-MBD transformed cells did not explicitly show such results. There wasn’t any major difference in bands between the induced and the uninduced samples. We believe this could potentially be because the MBD is an extremely
small domain of the protein and may be extremely hard to detect. Our strategy now would be to use more sensitive gels as well as separating the membrane fraction proteins from the rest of the cell for better results. At the same time, we have
begun testing how these engineered cells fare with lead samples.

### **Testing lead removal from artificially contaminated samples using ICP-MS** Inductively coupled plasma mass spectrometry (ICP-MS) is an analytical technique that can be used to measure elements at trace levels in biological fluids. We used this instrument, available in our institute, to test the concentrations of lead in our treated samples. Our engineered cells were induced using IPTG for different amounts of time and then exposed to Pb(NO3)2 solution such that the final concentration of lead in the sample was 400 ppm. The artificially contaminated samples were exposed to lead for 6 hours after which the solution was centrifuged to pellet the cells. Any remaining cells in the supernatant were removed using a filtration through a 0.2 micron filter. The concentration in the supernatant was checked using ICP-MS. This analysis were performed for both PbrR and PbrR-MBD transformed cells. ![](https://static.igem.wiki/teams/4428/wiki/result/results8.jpeg) ![](https://static.igem.wiki/teams/4428/wiki/result/results9.jpeg) Sample A represents the initial concentration of the artificially contaminated water. Samples B, C and D are the contaminated samples treated with PbrR-MBD expressing cells for 6 hours. The bacteria were induced for different durations before the treatment. Sample D is treated with uninduced cells: A. Original concentration of lead-contaminated sample, B. Contaminated sample treated with PbrR-MBD expressing bacteria which were induced for 6 hours, C. Contaminated sample treated with PbrR-MBD expressing bacteria which were induced for 12 hours (overnight induction), D. Contaminated sample treated with uninduced PbrR-MBD expressing bacteria. Sample A represents the initial concentration of the artificially contaminated water. Samples B, C and D are the contaminated samples treated with PbrR-expressing cells for 6 hours. The bacteria were induced for different durations before the treatment. Sample D is treated with uninduced cells: A. Original concentration of lead-contaminated sample, B. Contaminated sample treated with PbrR expressing bacteria which were induced for 6 hours, C. Contaminated sample treated with PbrR expressing bacteria which were induced for 12 hours (overnight induction), D. Contaminated sample treated with uninduced PbrR expressing bacteria. The observed results show a drastic decrease in the concentration of lead, for both, PbrR-MBD and PbrR expressing *E. coli.* This confirms that the engineered cells are able to substantially remove lead from the sample at this initial concentration of lead which is equivalent to that found in treated industrial wastewater. Additionally, we see 6-hour-induced samples perform better than overnight-induced samples which confirms the results from SDS-PAGE as well, which displayed the best expression of the protein at 6-hour induction. Interestingly, we observed that even the uninduced samples were able to treat the contaminated sample and remove lead from it. This was quite unexpected and led us to verify if there was leaky expression of the gene in the cell even in the absence of IPTG. This was verified by the study we referenced for the BclB anchor. S. Rangra et al. also used the same anchor protein under the control of a T7 promoter in the same strain we have used. Their results also reflect leaky expression of their cloned surface protein. Thus, we deduce that T7, being a strong promoter, does have some leaky expression in the cell due to which the uninduced cells are also expressing the lead-binding protein and adsorbing lead. Here, we believe that the concentration of lead provided in the sample is limiting the capacity of adsorption for the induced samples. Hence, we shall try to study the adsorption capacity of these cells at higher concentrations of lead in further experiments. **Testing lead recovery from engineered cells using ICP-MS** Since we observe the greatest amount of protein being expressed after 6-hour induction, we chose these 6-hour induced cells to test if it was possible to recover the adsorbed lead. We first used the engineered cells to treat artificially contaminated lead samples. Then, we centrifuged the sample to separate the cells from the treated water. The cell pellet was then treated with a strong acid, HNO3, to desorb the lead from the cell surface. The obtained HNO3 solution containing lead and cells was again centrifuged to remove cells out of it and the cell-free solution was tested for the recovered lead concentration using ICP-MS. ![](https://static.igem.wiki/teams/4428/wiki/result/results10.jpeg) ![](https://static.igem.wiki/teams/4428/wiki/result/results11.jpeg) We observe that almost all the lead adsorbed on the cells is recovered from the HNO3 treatment as we get extremely high concentrations of lead, greater than 390 ppb, after the desorption. The cumulative concentration after treatment and after recovery is observed to be slightly greater than 400 ppb which can be attributed to pipetting error. Additionally, we checked if the recovered cells underwent growth arrest or cell lysis after the HNO3 treatment for recovery. It was observed that the cells were still viable and didn’t undergo lysis. Thus, it was established that it is possible to recover most of the adsorbed lead through acidic leaching without lysing the cells.

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