iGEM 2023 — Team iit-delhi

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{% block title %}Notebook{% endblock %}
{% block lead %}This is our notebook of the whole project, in it you can find the progress we made each week from the initial brainstorming sessions, and up to the last stages. {% endblock %}
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### [Week 1](#collapse1)

The idea of the project, now called “Remedix” was discussed in the first week. Most of us were very new to the day-to-day working of an actual lab, and the transition from reading about it in books and papers to actually coming to work in a laboratory set up every day took a while to get used to. The first week we spent on getting well-versed with rather basic things like where the chemicals and equipment are located, and safety protocols to be followed.

### [Week 2](#collapse11)

Week 2 was spent learning basic microbiological techniques, preparation of enriched and minimal media for the growth of bacteria, sterilization techniques that are followed in the lab like how to safely operate the autoclave, streaking to obtain pure single colonies, maintenance of pure cultures, inoculations, making stocks of antibiotic solutions, filter sterilization..

### [Week 3](#collapse21)

We trained in basic molecular biological techniques like plasmid isolation, restriction enzyme digestion, learning to operate softwares to analyze a given DNA sequence which later helped us in designing unique and simple cloning strategies. We learnt how to set up Polymerase Chain Reactions (PCR) and extraction and purification of DNA from agarose gels. We learnt how to analyze different sizes of DNA on varying percentages of agarose gels, as well as learnt how to prepare different polyacrylamide gels to analyze protein samples of different sizes. We appreciated the fact that by changing the resolving gel percentage, we can visualize a lot of proteins across a wide range of molecular weights. Keeping the ultimate goal of driving our project, we placed the order for Bacillus licheniformis ATCC14580, as well as the primers needed to clone lichenysin.

### [Week 4](#collapse31)

We retrieved and revived glycerol stocks for the plasmids that we needed to clone lichenysin in, a plasmid called pSIM7 (of the recombineering plasmid tools, pSIM series (5676bp). We revived it by using chloramphenicol plates and media and we isolated the plasmid using the manual method described by Sambrook et al. It is a low copy number plasmid so the yield that we obtained for the first few times was very low. We had to repeat it thrice to get a good enough concentration to perform restriction enzyme digestion using SmaI and BglII for 1816 bp release. Following that, we had to optimize the amount of enzyme to use for this, depending on the concentration of the plasmid. We also performed gel extraction to obtain the linearized and digested plasmid, which we later would use as a vector to clone lichenysin..

### [Week 5](#collapse32)

We finally obtained Bacillus licheniformis ATCC14580 in a lyophilized form which we had to revive with UTMOST care and following stringent sterilization techniques. We noted the colony morphology, the time of growth. We performed colony PCR for the gene, lichenysin, from a freshly streaked B. licheniformis plate, with a long range polymerase, but we could not see any amplification of the desired size. So we tried putting the PCR again with the annealing temperatures across a wide range. This also did not yield any positive amplification corresponding to the desired size.

### [Week 6](#collapse2)

We planned and mapped out a different strategy to clone two different biosurfactants under two different promoters. We conducted an extremely exhaustive literature search to find suitable biosurfactants of two different compositions. We also looked into inducible, tightly regulated promoters so that we could produce a novel tunable system of biosurfactants. We finally narrowed it down to two biosurfactants, Alasan from Acinetobacter sp. and Rhamnolipid from Pseudomonas sp. to be cloned under the tac promoter and the tet promoter respectively. A detailed literature review was penned down elucidating why we chose these particular ones to work with.

### [Week 7](#collapse12)

To fasten up the process, instead of ordering the strains, we ordered the gene fragments for Alasan, aln, and rhamnolipid, rhlAB from iGEM sponsor IDT as well as designed and ordered primers to clone them in a plasmid system which already had the tac promoter cloned in between BglII and NdeI sites, called pTAC. We also obtained the plasmid construct having the gene fragment for Ptet to have everything ready in our hands before we dive into exhaustive cloning. Simultaneously, we also started screening the biosurfactants that are produced by other well known reported strains, like Pseudomonas sp., Franconibacter sp., Leifsonia sp., as well as Bacillus licheniformis. We performed EI assays, Oil displacement assays with cell free supernatant first. After that, we performed extraction of the biosurfactant by first doing lipid precipitation by HCl, and then extracting with methanol and chloroform.

### [Week 8](#collapse22)

We put our first test runs of the PCR reactions for Ptet, aln and rhlAB genes. Fortunately, we got desired amplification for all the inserts on the first go. We then proceeded to perform overnight digestion of these PCR purified products to have our inserts ready for setting up the ligation. During this week, we also decided to start optimizing the reaction conditions to prepare our three successive vectors for performing our three-step cloning. The restriction enzymes we were planning to use are NdeI, HindIII for cloning aln, BglII and SphI for cloning Ptet, and XbaI and SphI were cloning rhlAB. NdeI and HindII were relatively easier to optimize and we got a clean, single double digested vector in our second attempt with good yield even after gel extraction.
Fig 7. PCR amplified alasan (aln 1078 bp), 1kb ladder, Ptet (156 bp), rhlAB (700 bp) (left to right)

### [Week 9](#collapse33)

The troublemakers were the others, SphI and BglII. These enzymes are not compatible in the same buffer, and the release expected out of a double digestion of the plasmid, pTAC with these enzymes is only around 196bp, which was incredibly difficult to detect on the 1% agarose gel that we use normally. So, meticulously performing sequential digestion was our only choice. We first tried to linearize the plasmid with BglII, and then purify the linear band and use this elute to set an SphI enzyme digestion. After three attempts where we could not produce the desired results, we read on expert websites that we need to use the enzyme which needs a buffer with lesser ionic strength as that of the other enzyme. SphI was that enzyme for this set, so we wasted no time in setting up the digestion with SphI at first, making sure it gets linearized. We then performed PCR purification of the linear band on which BglII was added, after waiting for sufficient time a faint, diffused release of around 200bp was obtained. The final vector which we made required 2 more attempts.

### [Week 10](#collapse3)

The 10th week, we set up our first ligation reaction to clone Alasan, aln, with our vector, pTAC digested with NdeI and HindIII. We used 1:3 molar ratios of vector: insert and used T4 DNA ligase, following transformation into freshly prepared chemically competent cells of E. coli DH5α. The next day we performed colony PCR to check the presence of recombinants in the cells, and we found two of them were positive for the clone. We inoculated these two colonies to perform plasmid isolation the next day and to perform a PCR against the gene aln with the plasmids as well as confirm the clones by restriction digestion. However, we failed to see desired amplification for aln and the insert released after the restriction digestion of these plasmids were also of the wrong size. We set up ligation for alasan, aln two more times using various other vector preparations and various other molar ratios of inserts and vectors but we failed to get a clone all these times.

### [Week 11](#collapse13)

Since we were struggling with the cloning for alasan, we decided to start attempting to clone Ptet. We got that clone in two attempts and confirmed the clone by plasmid PCR and digestion by XbaI (a non-cutter we included in our primer sequence). Simultaneously, from our troubleshooting sessions with our mentor, Prof. Preeti Srivastava, we hypothesized that probably our aln insert is not getting properly digested due to which it is hindering the process of efficient ligation. We then decided to perform ligation of our Taq polymerase amplified insert aln as well as rhlAB pGEM®-T Easy Vector, and perform a rapid ligation. The next day we screened our recombinants using blue white screening, wherein we selected the white colonies to proceed for plasmid isolation and restriction digestion. The digested inserts were then used for ligation.

### [Week 12](#collapse23)

On our 4th attempt at ligation with our previously prepared insert, we got the clone for alasan. We checked its expression in whole cell lysate, sonicated pellet and sonicated supernatant (to check if the protein is being able to be properly folded; hence solubilized) on a 12% SDS-PAGE. We then immediately proceeded to do preliminary biosurfactant assays with different types of oils, like the emulsification index, to confirm the presence of alasan. After these assays gave us positive results, we extracted the biosurfactant and prepared its crude extract. To solubilize alasan, we checked various solvents and then observed the best solvent was deionized water. To validate our recombinant biosurfactant, we ran a simple qualitative analysis to determine its components on Thin Layer Chromatography, which gave us indication of proteinaceous and polysaccharide moieties in Alasan, which completely correlated to what we had previously read in literature.

### [Week 13](#collapse34)

Now that a preliminary TLC has been conducted which already validated our recombinantly produced alasan, we went further in this path to throw light on the functional groups that can be detected in the biosurfactant through Fourier Transform- Infrared Spectroscopy. The transmittance vs. wavenumber spectra helped us to assign peaks corresponding mainly to primary and secondary amines, on C=O bonds and also alcohol and carboxylic acid bonds. This further correlated to what we know of alasan to be of proteinaceous and polysaccharide in nature.

### [Week 14](#collapse35)

On the Ptet-rhlAB cloning front of our work, we attempted three trials of ligation to clone rhlAB downstream to Ptet. After it failed multiple times, we again hypothesized that the insert and the vector might not be getting properly digested, so we incubated them with the enzymes for a longer period of time to get another set of purified inserts and vectors for ligation.

### [Week 15](#collapse14)

On the Ptet-rhlAB cloning front of our work, we attempted three trials of ligation to clone rhlAB downstream to Ptet. After it failed multiple times, we again hypothesized that the insert and the vector might not be getting properly digested, so we incubated them with the enzymes for a longer period of time to get another set of purified inserts and vectors for ligation.

### [Week 16](#collapse24)

We had read a plethora of research as well as review articles on many peer reviewed journals where scientists have documented and demonstrated that biosurfactants have the incredible ability to trap, or “cage” heavy metals, like lead, cadmium, copper, nickel, chromium etc. Now that we had our hands on the extracted alasan biosurfactant, we wanted to see how active it is to trap some heavy metals that are reported to be notorious for ecological welfare. We performed ICP-OES (Inductively coupled plasma- Optical EMission Spectroscopy) with appropriate controls with metals like lead, copper, cadmium, chromium, and nickel to see if the recombinantly expressed alasan is being able to cater to this particular need. To the best of our knowledge, the action of alasan on heavy metals such as these have actually not been reported in the community. We then held two sessions with our investigator, Prof. Preeti Srivastava who guided us with a proper experimental set up to test our hypothesis.

### [Week 17](#collapse36)

After conducting ICP-OES, we tabulated our findings.we also started compiling our results. We also tried genomic DNA amplification of Bacillus licheniformis with success. We will be attempting to brain the gene for lichenysin by PCR again, to clone in our already prepared pSIM7 vector.

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{% block lead %} iGEM-IIT-Delhi page for engineering and design.
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JSON Animation Example

## Introduction

bioSURF is a project aimed at using biosurfactants to rid the environment of the heavy metals that pollute every ecological niche around us, owing primarily to industrial discharge. Biosurfactants are non-toxic, biodegradable, surface-active agents that find a variety of applications. However, their production in the industry faces certain major limitations. The main issue with the production of biosurfactants is that the wild strain that produces them is often pathogenic.They have been produced only in low yields in the industry and have a very high incubation time. They must also be separated from the media their hosts were cultured in, which leads to steep purification costs. This, despite their numerous benefits, renders them economically unviable.

Most research and development related to biosurfactants aims to overcome these challenges by utilizing GMOs for their production.

## LOOP1

### DBTL1

D: A literature review determined that Lichenysin, the biosurfactant with the greatest chelating power, has never before been produced in a model organism. So we decided to undertake the task ourselves, by genetically modifying E. coli to produce Lichenysin. We planned to insert the 26.6kb cluster lchAA-AB-AC-TE in the plasmid PSIM7, which we would then introduce into E. coli.

B: We planned to achieve this by amplifying the entire 26.6kb insert, using Fantom polymerase. This would allow us to amplify such a large sequence. We had also designed primers with overhang to facilitate specific binding.

T: The PCR results were obtained by means of 1% Agarose gel electrophoresis, which yielded too many bands. This indicated non specific amplification and implied our current method needed improvement.

L: Numerous experts we interviewed pointed out that incorporating such a huge insert into a plasmid in one go is not a viable plan. Hence, attempted a different method.

### DBTL2

D: We had to ideate a new plan to counter the non-specific amplification observed in our first cycle. We decided to split our insert into 5 fragments and incorporate them by means of a Gibson assembly.

B: Primers had to be designed for all 5 fragments and the insert was added to the vector and amplified by PCR.

## LOOP2

### DBTL1

D: In our journey of trying to clone Lichenysin, we learnt a lot about different biosurfactants and their varied properties and applications. We realized that oftentimes, a mixture of biosurfactants is desired. Hence, we decided to attempt to create a tunable, bidirectional promoter for the controllable production of the biosurfactants Alasan and Rhamnolipid, which is a result that has never been reported in literature! This enables us to produce two different biosurfactants, using a plasmid of just 5.7kb. This causes considerably lower metabolic load on the GMO, especially in comparison to our previous design, in which the insert itself was 26.6kb. Also, as a mixture of 2 different biosurfactants is obtained, this has much lower purification costs associated with it. Moreover, the biosurfactants are inducible, the addition of IPTG [Isopropyl ß-D-1-thiogalactopyranoside] yields Alasan while Doxycycline triggers Rhamnolipid production by activating Ptet promoter. Ptet is a constitutive promoter that is repressed by TetR. When Tetracycline derivatives such as Doxycycline bind to it and remove the TetR, it shows promoter activity. Hence it is inducible and its expression is tightly controlled. This inducibility is advantageous as it implies that we do not need to wait for the onset of the stationary phase, which takes about 3 days, the production of biosurfactant starts instantaneously. This system also entirely bypasses the need to have the clone expressing in a specialized protein expression strain, like the BL21 (DE3) because we are cloning the gene for Alasan under a tac promoter, not a T7 one.

B: The plasmid used to realize this schematic was pTAC, which has a Ptac promoter. However, for us, it only had 4 other viable sites due to resource constraints: BglII, SphI, HindIII and NdeI. We needed to add the insert Aln, for Alasan production from the promoter Ptac, which was already a part of the plasmid. We also needed to incorporate the promoter Ptet and the insert rhlAB for Rhamnolipid production. This roadblock was overcome by designing the primer for Ptet with an overhang that contained the site for XbaI, thereby securing another site to facilitate the addition of all desired sequences.
However, we ran into another issue, as we discovered that the enzymes BglII and SphI were not compatible with the buffer used in the restriction digestion of HindIII and NdeI. This was resolved by sequential digestion. The plasmid was first digested with SphI (followed by PCR purification once it linearized), after which we added BglII to the purified elute to get a release of around 200bp. E. coli was then inoculated with the modified plasmid, containing Ptac, Aln, Ptet and rhlAB.

T: We were facing quite a few issues regarding the success of our ligations. Even after the concentrations of inserts and vectors were at par, we were not able to obtain the clones even after 3 iterations.

L: We hypothesized that our PCR purified inserts, aln and rhlAB, are not properly getting digested with the respective enzymes, NdeI and HindIII for aln and XbaI and SphI for rhlAB. This seemed to be the only possible conclusion as the insert digestion is the only thing that is difficult to verify.

### DBTL2

D: So as to tackle the aforementioned problem, we came up with an alternate strategy to get 100% digested inserts by means of cloning our PCR products in a pGEM®-T Easy Vector. We strategize that as the inserts were amplified using Taq polymerase, TA cloning can be performed for their incorporation in the pGEMT plasmid. After this the cloned colonies obtained through blue white screening can be used to obtain plasmids. These can be digested further and a clear release would be visible to verify success of the experiment.

B: We used our Taq polymerase amplified inserts to clone in the vector, performed rapid ligation for only one hour at 37 degrees Celsius, followed by transformation of the mixture in chemically competent E. coli DH5α cells and screened our positive recombinants using blue white screening. The white colonies after ligation and transformation were then used to carry out plasmid isolation after which we carried out restriction digestion to see the accurately sized insert fall out and confirm the clones.

T: Properly digested release of rhlAB and aln were obtained which were again used for setting ligation. Alasan cloning was successful but rhlAB still didn't bore a result.

![Image Description](https://static.igem.wiki/teams/4898/wiki/self-legation.jpeg)

L: We had to repeat this loop multiple times as this was an incredibly difficult cloning to achieve. We had to optimize the insert and vector molar ratios for the perfect ligation mixture which finally yielded our desired insert, after many repeated trials and attempts to digest the vector and insert. As mentioned before, the pGEMT cloning came in handy during our dire need to have perfectly digested inserts. But, we still couldn’t achieve the bidirectional promoter in a single plasmid, as we were hindered by two of our sites: XbaI and SphI being only 4bp apart, which made this extremely susceptible for self ligation of the vector.

### DBTL3

D: This time we decided to set the ligation with a control to check for self ligation.

B: We prepared two ligation mixtures, one with the insert and the other without it. They were then ligated and transformed.

T: As expected the control bore a comparable number of colonies to the one with the insert concluding that self ligation is preventing final cloning.

![Image Description](https://static.igem.wiki/teams/4898/wiki/somebluewhite.jpg	)

L: Further optimisation of insert and vector concentrations would be required to obtain the clone.

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