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