Key

Transformation

Design
Preparation of Competent E. coli TG1 cells using CaCl2 solution and Heat shock Transformation with GFP plasmid.

Build
Link to protocol for transformation.

Test
Plating of transformed and non-transformed control bacteria in ampicillin plates. Successful Transformation was achieved. However, the plates have lawns.

Learn
The transformation efficiency seems to be quite high and hence the incubation or/and the volume of solution must be reduced.

Design
Same Protocol, with lower incubation and halving the volume of transformant cells to be plated.

Build
Same as mentioned in the above cycle.

Test
Plating of transformed and non-transformed control bacteria in kanamycin plates. Individual colonies of transformed bacteria were obtained.

Design
Preparation of competent cells with MgCl2 at first suspension and CaCl2 in the second suspension. Transformation as usual.

Build
Same as mentioned in the above cycle.

Test
Plating of transformed and non-transformed control bacteria in kanamycin agar plates. Successful, with marked increase in the number of single colonies.

Protein Expression

The gene of interest (anti-IL-8) in the plasmid is regulated by the lac operon. IPTG (Isopropyl β-d-1-thiogalactopyranoside), a molecular mimic of allolactose can act as the promoter and is often used because it is not metabolized but as a con, it also has a toxic effect on cells.

Design
IPTG Induction of transformed bacteria.

Build
1mM concentration of IPTG was used to induce E. coli BL21 transformed with GFP AmpR plasmid, cultured to mid-log phase.

Test
The cells are pelleted down, and the supernatant is used to run a western blot (Since the protein is extracytoplasmic). Low expression observed.

Learn

• IPTG concentration is not enough.
• IPTG is not the best inducer in this case.
• The stock IPTG is too old to promote good enough expression.

Design
Induction with lactose (skimmed milk) instead of IPTG.

Build
Use Skimmed Milk Broth (1% peptone + 1% NaCl + 0.5% Yeast Extract + 1% Skimmed milk powder in MilliQ water) as a culture medium cum inducer - since allolactose can directly induce lac operons.

Test
Curdling of Milk is seen when autoclaved.

Learn
Skimmed Milk broth can be sterilized after preparation; we have to prepare the media in a sterile environment.

Design
Skimmed Milk broth/ Lactose Induction

Build
Autoclave LB solution and add 1% sterile skimmed milk powder.

Test
The cells are pelleted down, and the supernatant is used to run a western blot (Since the protein is extracytoplasmic), which showed almost negligible induction.

Learn
Skimmed Milk is not a good promoter.

Design
We try a gradient of IPTG concentrations on different cultures to optimize induction. Fresh IPTG stocks (100mM) were procured.

Build
Link to Optimization of Expression under protein expression in experiments.

Test
The cells are pelleted down, and the supernatant is used to run a western blot (Since the protein is extracytoplasmic) which shows sufficient induction at 1mM.
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Design
We now varied the temperature of final incubation to optimize the temperature requirements of our plasmid.

Build
Prepare cultures, induce with 1mM IPTG, and incubate for 12-14 hours at 10°, 15°, 20°, 25°, 30°, 37°C.

Test
We again ran a western blot with all types of supernatants and got the strongest signal at 25°C.

Learn
The optimized conditions for our plasmid to produce proteins in bacterial BL21 chassis is 1mM IPTG at 25°C at 230 rpm.

OIL8 Plasmid Strain Selection

Design
We compared TG1 and BL21 strains of E. coli to choose a chassis for protein expression. Both were transformed with OIL-8 plasmid.

Build
We induced transformed cultures of both strains with the same concentration of IPTG at the same conditions.

Test
BL21 showed considerably high expression in the Western Blot with cell lysates from both cultures.
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Learn
BL21 was chosen as the chassis for protein production.

Plasmid Extraction

Design
Kit-free Extraction to isolate plasmid from E. coli DH5-alpha bacteria containing OIL-8 plasmid (provided by AddGene).

Build
Link to Alkaline Lysate Protocol .

Test
No pellet was left over at the end of the protocol.

Learn
The isopropanol necessary to precipitate the plasmid is light-sensitive, also the reason why it is kept in colored bottles. So, the very old stocks we had were possibly quite ineffective.

Design
Same as before, with new isopropanol procured from the chemistry lab.

Build
Same as mentioned in the above cycle.

Test
No pellet is obtained, leading to a search for better procedures since our experience meant we didn’t have optimally pure reagents or conditions necessary to carry out the process with enough precision to give us a workable yield.

Design
We resort to using ThermoFisher MiniPrep Kit for plasmid extraction.

Build
Link to MiniPrep Kit Protocol.

Test
Successful, with viable concentrations under Nanodrop - about 25µg/mL.

Protein Expression for Anti-IL8

Design
Since this was a secreted protein, buffers used in training runs wouldn't have good efficiency with the exact same composition. So, we needed the buffer selection runs.

Build
pI= 5.7 → pH > pI
Trial Buffers: Phosphate (pH=8), Tris Cl (pH=8), PBS (pH=7.4)
Additives: PMSF [Protease Inhibition], β-mercaptoethanol [Reducing Agent], Glycerol [10%]
Comparison: Yield [Nanodrop], Purity [SDS Page], Stability [Aggregation]
Salt: NaCl

Test
We ran SDS PAGE's with 1. Varying buffers, 2. Varying salt concentrations

Learn
Unclear data. Salt concentration chosen 300mM.

Design
Western Blot.

Build
We run the cell lysate to see if the protein is produced.

Test
The gel is successful: visible bands are formed at 31kDa.

Learn
Protein is successfully produced by plasmid.

Design and Build
We decided to carry out ELISA with our Antibody as a secondary and IL8 as primary, using HRP conjugation to quantify the interactions between IL8 and O-IL8-15.

Test
Unsuccessful, with no color changes.

Learn
Ineffective protein folding might be the reason for failure. So, we looked into literature, and concluded there could be two issues: one, disulfide linkages not being formed properly; two, protein being secreted in the form of Inclusion Bodies.

Design
Ammonium Sulphate Precipitation to concentrate supernatant.

Build
Link to ammonium sulphate precipitation protocol.

Test
Ran the above at varying concentrations to find the optimum saturation.

Learn
70% saturation was found to be best.

Design
BLI with Tris Cl as a buffer.

Build
BLI gives us a quantitative measure of the binding affinity of IL8 and Anti IL8.

Test
We get unclear data.

Learn
The buffer might have led to improper binding. Hence, we change the buffer to PBS.

Design
BLI with PBS as buffer.

Build
Quantitative test of binding affinity of IL8 and Anti IL8.

Test
Worse binding compared to Tris-Cl.

Learn
Ineffective folding might be the issue. We changed our buffer back to Tris-Cl. All further steps used Tris-Cl pH 7.4, unless mentioned.

Design
Urea precipitation to check presence of protein in IBs.

Build
Link to urea precipitation protocol..

Test
SDS-PAGE showed no presence of protein in purified pellet.

Learn
Protein may not have been properly produced, due to the lack of formation of disulfide linkages. Should either use shuffle cells or target protein to the periplasmic space using PelB signal peptide.

Design
Switch to SHuffle Cells. We transfect SHuffle Cells using the original plasmid as well as we have. These cells are designed to produce necessary disulfide linkage as and where required, thus solving our issue.

Test
To Be Done

Design
We designed a new plasmid with PelB signaling peptide instead of OmpA.

Build
We intend to transform the chassis with the modified plasmid to send the protein first to the periplasmic space, to render the necessary disulfide bonds.

Test
To Be Done

Lipid Nanoparticle Formulation and Characterization

Design
LNPs with the below composition were fabricated using the iLNP chip at flow rate ratio (mRNA:lipid) =3:1 i.e 240 and 80 ul/min using a 20 baffle mixer. Nishant, a PhD mentor, suggested that we use a 20 baffle mixer (instead of 30 or 10) since they were pre-optimised for 100nm LNPs. Based on our inputs from Dr. Pradipta, the LNPs were formulated with four lipids: ALC-0315, mal-PEG 2000, cholesterol and DSPC. ALC-0315 was the cationic lipid. It was necessary as RNA is negatively charged and the cationic lipid helps in the uptake of the RNA. The molar ratios used were ionizable cationic lipid: neutral lipid: cholesterol: PEG-ylated lipid:: 50: 10: 38.5: 1.5. DSPC is the neutral lipid.

Build
Link to protocol.

Test
DLS (Dynamic Light Scattering) analysis of the first LNP batch yielded results showing a radius of around 180nm.

Learn
Tested LNPs are close to required characteristics. Nishanth, who helped us in this process, advised us to redo the fabrication of the LNPs at high speed to reduce the LNP sizes to near about 100nm.

Test
Second run of DLS yielded LNP sizes of about 500nm.

Learn
The high speeds used could have damaged the microfluidic device, resulting in a larger average radius due to improper mixing. We hypothesized the cause for the radius mismatch as lack of information and optimization of the speed necessary for the LNP.