iGEM 2022 — Team stony-brook

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![](https://static.igem.wiki/teams/4235/wiki/real-collage.jpg)

[Team Notebook](https://docs.google.com/document/d/1NGmnna2cduqIWc0kMPd_BLLp59GXX6ZEntLpcpgYY-c/edit?usp=sharing)

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# Engineering Success

Synthetic biology is widely considered to be a discipline which involves the engineering and development of new biological systems and devices, or the redesigning and improvement of current systems. Engineering widely refers to the process of designing, constructing, and building something. The engineering process is critical to success. Synthetic biology in particular, widely relies on the engineering cycle: DBTL; Design, Build, Test, and Learn to successfully innovate, develop, and implement novel biological systems.

This past summer we initiated the production of our product, PROS. We laid the basic groundwork for a future therapy for protein S deficiencies. Our goal for the summer was to successfully express human recombinant protein S in SF9 insect cells and *E.coli*. In order to accomplish this, we applied the DBTL cycle numerous times. As a team of 11 undergraduate students, the engineering cycle was critical to our success. Although we faced various obstacles and challenges, this process helped us persevere and succeed in our project. On this page we detail our iterations of the design cycle, and how it helped us build and refine our project.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/gray-simple-cycle-diagram-chart.png)

---

### Overall Design

In our project, we decided to try and express our protein of interest, protein S, in two different organisms: SF9 insect cells, and *E.coli*. We wanted to compare prokaryotic vs. eukaryotic expression, and to this extent, we conducted experiments in parallel. Although we could not conduct the SF9 experiments to completion, both of these aspects of our project underwent the engineering process numerous times.

![](https://static.igem.wiki/teams/4235/wiki/real-engineering-success/screen-shot-2022-10-09-at-3-10-27-pm.png)

---

### Engineering Cycle for *E.coli*

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/dbtl-e-coli.jpg)

## *Cycle 1: Obtaining Pure Cloning Substrates for the Ligation Independent Cloning Reaction*

**Design:** Our aim was to insert the PROS gene, which was codon optimized for chassis *E. coli* and obtained through synthesis from IDT into the pET His6 LIC cloning vector (2Bc-t). In order to prepare our plasmid for the LIC, we went through several cycles of minipreps, linearization using the restriction enzyme Hpa1 and gel extractions in order to get high quality substrates for the cloning reaction. Our Gene of Interest was amplified using the primers we designed, creating specific overhangs which would facilitate the annealing step during LIC. More detailed information on our primer design and LIC process can be found on our [Design page](https://2022.igem.wiki/stony-brook/design).

**Build**: For preparing our GOI and the plasmid for the LIC reaction, we performed a T4 polymerase 3’ chewback process by treating the insert with dGTP and T4 polymerase and the vector with dCTP and T4 polymerase. The 3’ chewback would generate complementary 5’ overhangs which would facilitate the annealing reaction. GOI concentration = 136.7 ng/uL Vector concentration = 7.2 ng/uL

**Test**: After generating the substrates for LIC with 5’ overhangs, we performed the following annealing reactions:

Experimental sample: 10 uL GOI + 5 uL vector + 5 uL Molecular grade H2O (Final vol of 20 uL)

Negative Control sample: 5 uL vector + 15 uL Molecular grade H2O (Final vol of 20 uL)

We transformed the annealing reaction products in *E. coli* DH5 alpha using 10 uL of the 20 uL samples and incubated the cells at 37 °C for 1 hour, shaking at 220 rpm. Transformed cells from both samples were plated on ampicillin agar plates and incubated overnight.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/aug-4-2022.jpg)

***Figure 1.*** Transformation plate of the experimental sample
![](https://static.igem.wiki/teams/4235/wiki/engineering-success/aug-4-2022-positive-control.jpg)
***Figure 2.*** Transformation plate of the negative control

**Learn:** No colonies were observed on the experimental control plate, indicating that the LIC reaction was successful. We thought that our plasmid samples had very low concentrations for the annealing reaction and the ratio of Insert to vector might be low. We decided to perform more minipreps to obtain higher concentrations of the plasmid and reattempt the LIC reaction.

*## Cycle 2: Obtaining Higher Concentrations of the Plasmid and Reattempting the LIC Reaction*

**Design:** To improve the yield of our plasmid samples, we performed another miniprep. We obtained multiple samples of the mini prepped samples to increase the chances of obtaining high quality LIC substrates after going through the steps of linearizing and gel extraction. Moreover, the concentration of the plasmid decreases significantly after going through the processes of restriction digest and gel extraction. After treating all the samples and analyzing them using gel electrophoresis, we concluded that one of the mini prepped samples was concentrated and pure enough for the Ligation Independent Cloning reaction.

**Build:** Final concentrations of our GOI and the plasmid after preparing the LIC samples were 68.35 ng/uL and 5.55 ng/uL respectively. We decided to perform two separate annealing reactions using 2 different insert to vector ratios, 1:5 and 1:2.

Experimental Reaction 1: 2 uL GOI + 10 uL vector + 8 uL Molecular grade H2O (Final vol of 20 uL)

Experimental Reaction 2: 5 uL GOI + 10 uL vector + 5 uL Molecular grade H2O (Final vol of 20 uL)

**Test:** We transformed the annealing reaction products in *E. coli* DH5 alpha using 10 uL of the 20 uL samples and incubated the cells at 37 °C for 1 hour, shaking at 220 rpm. Transformed cells from both the reaction samples were plated on ampicillin agar plates and incubated overnight.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/aug-11-2022-experimental-plates.jpg)

***Figure 3.*** Transformation plates of DH5alpha

**Learn:** We observed colonies on both the transformation plates, indicating that both the experimental reactions for Ligation Independent Cloning were successful. After the successful cycle of designing, building, learning and testing for optimizing our experimental conditions to create our recombinant plasmid, we decided to conduct confirmatory experiments like restriction digests, PCR amplification of the insert from the recombinant vector and sequencing the plasmid sample. We were able to conclude that our GOI was successfully cloned into the plasmid by analyzing the results of all 3 confirmatory tests. More detailed information on our experiments can be found on our [Results page](https://2022.igem.wiki/stony-brook/results).

## *Cycle 3: Conducting Expression Tests to Detect Recombinant Protein S Expression*

**Design:** After confirming the presence of our GOI in the plasmid, we transformed the recombinant vector in *E. coli* strains BL21 and Origami B cells. BL21 is the common lab strain used for expressing recombinant proteins. However, BL21 cells are not very efficient in making disulfide bonds in the expressed protein, making it difficult to express soluble protein. Therefore, we decided to conduct parallel expression tests in both Bl21 and Origami B cells. Origami cell strain contains specific mutations which greatly enhance their ability to make disulfide bonds in the expressed proteins, increasing protein stability and preventing degradation.

**Build:** For the expression test, we picked colonies containing our recombinant vector, cultured them in 5 mL LB and ampicillin overnight at 37°C, and used 125 mL of the overnight culture to dilute it into a 25 mL culture. The 25 mL cultures of both BL21 and Origami B were grown to OD600 at 37°C and induced using 1 mM IPTG.

**Test:** We collected a 1 mL pre-induction sample from both the BL21 and Origami B cultures before adding 1mM IPTG. After induction, the cultures were grown for 4 hours at at 37°C and 220 rpm, and a post-induction sample was obtained every hour post-induction for 4 hours. All the expression samples were centrifuged at 3000 rpm for 5 min, to harvest the cells in the pellet.
We performed an SDS-PAGE and a Western Blot to detect expression.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/results-of-sds-page.jpg)

***Figure 4.** SDS-PAGE of the BL21 and Origami B expression samples.*

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
||  |  |
| --- | --- |
| Well # | Sample |
| Well 1 | Ladder |
| Well 2 | Origami B - Pre-Induction |
| Well 3 | Origami B - 1 hr Post-Induction |
| Well 4 | Origami B - 2 hr Post-Induction |
| Well 5 | Origami B - 3 hr Post-Induction |
| Well 6 | Origami B - 4 hr Post-Induction |
| Well 7 | BL21 - 1 hr Post-Induction |
| Well 8 | BL21 - 2 hr Post-Induction |
| Well 9 | BL21 - 3 hr Post-Induction |
| Well 10 | BL21 - 4 hr Post-Induction |

***Table 1.*** SDS-PAGE well setup

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/results-of-western-blot.jpg)

***Figure 5.*** Western blot displaying the first expression test result.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
||  |  |
| --- | --- |
| Well # | Sample |
| Well 1 | Ladder |
| Well 2 | Origami B - Pre-Induction |
| Well 3 | Origami B - 1 hr Post-Induction |
| Well 4 | Origami B - 2 hr Post-Induction |
| Well 5 | Origami B - 3 hr Post-Induction |
| Well 6 | Origami B - 4 hr Post-Induction |
| Well 7 | BL21 - 1 hr Post-Induction |
| Well 8 | BL21 - 2 hr Post-Induction |
| Well 9 | BL21 - 3 hr Post-Induction |
| Well 10 | BL21 - 4 hr Post-Induction |
| Well 11 | Ladder |

***Table 2.** Western blot well setup.*

**Learn:** The results of SDS-page were inconclusive because we did not observe any obvious dark bands at about 70 kDa (approximate weight of protein S), and all the bands on the gel are roughly of the same intensity, indicating no increase in expression with time. The Western Blot results revealed that BL21 cells are unable to express protein S, as no bands were observed for BL21 samples. Origami cells, however, did show bands at around 25 kDa, indicating that protein S is being degraded after expression. We thought that the conditions we used for inducing these cells were not favorable and were causing the protein to form aggregates in the cells. Therefore, we decided to brainstorm the ways in which we could optimize the expression conditions for Origami B cells.

*## Cycle 4: Optimizing Expression in *E. coli* Origami B Cells*

**Design:** In the previous expression test, we observed expression of protein S from Origami B cells, and the fact that BL21 were unable to express protein S indicates that Origami B cells are better suited for expressing protein S because of their ability to make disulfide bonds in the protein. We decided to perform another expression test in Origami B cells by manipulating several experimental conditions. We decided to induce two different samples of Origami cells, each at a different cell concentration (OD 0.4 and 1.1). We also decreased the concentration of IPTG from 1 mM to 0.5 mM for induction and the origami cells were induced at 37°C, gradually reducing it to 23°C. Moreover, we decided to sonicate our cells before conducting the western blot and adding protease inhibitors.

**Build:** We picked 2 colonies from the Origami B transformants and cultured them overnight in 5 mL of LB. Both samples (Ori 2 and Ori 3) were diluted into 25 mL cultures and sample Ori 2 was induced at OD = 0.4 and sample Ori 3 was induced at OD = 1.1. We collected one pre-induction sample, 4 post-induction samples (one hour increments for 4 hours) and one overnight induction sample.

**Test:** After sonicating the cells in our expression samples and addition of protease inhibitors, we performed a Western Blot for detecting expression of protein S.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/western-blot-second-cycle.jpg)

***Figure 6.** Western blot of the second expression test.*

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
||  |  |
| --- | --- |
| Well # | Sample |
| Well 1 | Pre-induction Sample (Ori 2) |
| Well 2 | One hour post-induction (Ori 2) |
| Well 3 | Two hours post-induction (Ori 2) |
| Well 4 | Three hours post-induction (Ori 2) |
| Well 5 | Four hours post-induction (Ori 2) |
| Well 6 | Overnight induction (Ori 2) |
| Well 7 | Empty well (loading dye) |
| Well 8 | Pre-induction Sample (Ori 3) |
| Well 9 | One hour post-induction (Ori 3) |
| Well 10 | Two hours post-induction (Ori 3) |
| Well 11 | Three hours post-induction (Ori 3) |
| Well 12 | Four hours post-induction (Ori 3) |
| Well 13 | Overnight induction (Ori 3) |

***Table 3.** Western blot well setup.*

**Learn:** As evident from Figure. we were successfully able to detect protein S expression (full length, about 70 kDa) using the HRP anti-His 6 antibodies. We learned that the ability of Origami B cells to make disulfide bonds greatly enhances protein stability, making it possible for us to express recombinant human protein S in a bacterial cell strain. We also learned that some of the protein was still being degraded, as evident from the multiple bands visible from about 30-50 kD. We thought that the protein is being degraded from the N-terminal because human protein S has multiple N-linked Glycans added during its post-translational modification. Glycans might have protective functions for the EGF domains on the protein. As we successfully expressed protein S in its full length we thought that the conditions for expressing recombinant protein S in bacterial cell strains can be further optimized.

## *Conclusion*

After going through a lot of cycles of designing, building, testing and learning, we were successful in generating the recombinant plasmid containing the PROS gene and expressing protein S in its full length by optimizing expression in Origami B cells. After concluding our wet lab experiments, we started thinking about ways in which future igem teams can further improve recombinant protein expression in bacterial cells. We also partnered with the UCSC igem team and they helped us model the expression of protein S in yeast, which, being an eukaryotic organism, is a promising host choice for protein expression. After conducting a literature search and consulting with our advisor Andrew Sillato, we came to a conclusion that expressing recombinant protein S along with glycosyltransferases in both E. coli Origami B and yeast cells is a promising future avenue for protein S research.

---

### Engineering Cycle for SF9

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/dbtl-sf9.jpg)
![](https://static.igem.wiki/teams/4235/wiki/engineering-success/red-graph.jpg)

## *Cycle 1: Determining Appropriate Cloning Technique and Primer Design*

**Design:** Our goal was to clone the PROS gene which was codon-optimized for chassis SF9 and obtained through synthesis from IDT, into the multiple cloning site of the Baculovirus transfer vector - pFastBac YMBac-II. After a lot of literature review and consultation with our advisor, we determined that Ligation Independent Cloning would be the ideal technique for our cloning process. We designed primer sequences that would be used to amplify the synthesized PROS gene and the plasmid pFastBac, in order to generate specific overhangs on the cloning substrates. Detailed information regarding our primer design and the LIC cloning process can be found on our [Design page](https://2022.igem.wiki/stony-brook/design).

**Build:** The building phase for our first cycle of engineering involved obtaining our synthesized gene of interest, plasmid and the designed primers and amplifying the DNA sequences using PCR. The annealing temperature for our PCR reaction was calculated using the [NEB Tm calculator](https://tmcalculator.neb.com/#!/main) and the thermocycling conditions were set using the [NEB PCR protocol for Q5 Polymerase](https://docs.google.com/document/d/1CsBfUaFsfK5yVQ1qeWmfUk5dveblDTBFh2gpRXSQJ4A/edit?usp=sharing).

Annealing step (both GOI and plasmid): 55°C for 30 seconds
Extension: 72°C for 2 minutes (30 sec/kb)
Final Extension: 72°C for 2 minutes

**Test:** We ran the PCR reaction for both the Gene of interest and the plasmid at same thermocycling conditions and analyzed the results using gel electrophoresis on a 1% agarose gel.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/july-1-2022.jpg)

***Figure 7.** 1% agarose gel of the insert and plasmid PCR products. Well 3: DNA ladder, Well 4: Gene of interest PCR product, Well 5: Plasmid PCR product*

**Learn:** We were able to amplify our GOI (~2031 bp) and the plasmid (~5000 bp). However, we learned that these specific thermocycling conditions resulted in significant DNA laddering for the plasmid sample as evident from Figure 1, which can be a result of nonspecific primer annealing. Moreover, the smearing on the gel can be a result of using too much DNA substrate for PCR, high concentrations of the primers or the enzymes or contamination. We also attempted performing the cloning reaction using these products, however, it was unsuccessful. Therefore, we decided to optimize the conditions for our thermocycling reaction and re-attempt PCR amplification.

## *Cycle 2: Optimizing PCR Thermocycling Conditions for the Plasmid*

**Design:** We decided to improve our PCR conditions and conduct another thermocycling reaction for amplifying our plasmid using annealing temperatures of 55°C and 62°C. We thought that increasing the annealing temperature for one of the thermocycling reactions might lower the nonspecific annealing of the primers, and using the same initial annealing temperature of 55°C would serve as a control and comparison with the initial PCR reaction. We also decided to perform a gel extraction of the PCR products to obtain more pure samples for attempting the cloning reaction.

**Build:** We made two different PCR substrate samples for the thermocycling reactions at 55°C and 62°C and increased the extension time from 2 to 2.5 minutes and the final extension time from 2.5 to 5 minutes.

**Test:** We ran the PCR reaction for both the thermocycling conditions and analyzed the results using gel electrophoresis on a 1% agarose gel.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/july-14-2022.jpg)

***Figure 8.** 1% agarose gel of the plasmid PCR products. Well 1: DNA ladder, Well 2: Plasmid Tm = 55°C Well 3: Plasmid Tm = 62°C*

**Learn:** By trying a different thermocycling condition, we were able to reduce some of the DNA laddering and gel smearing. However, there were still some non-specific bands for both the samples and annealing temperatures. We performed a gel extraction of the PCR products and attempted another cloning reaction, which was again unsuccessful. We also realized that for all our prior PCR attempts, we used the primer overhang sequences for calculating the annealing temperatures. Therefore, for the next cycle of optimizing PCR conditions, we used the entire primer sequence, i.e. both the overhang sequences and the overlapping sequence.

## *Cycle 3: Optimizing PCR Thermocycling Conditions for the Plasmid and the GOI*

**Design:** Since the previous cycles of optimization were only able to slightly reduce DNA laddering for the plasmid PCR products and the consequent cloning reactions failed, we decided to use the entire primer sequence for calculating the annealing temperature and attempt another PCR reaction.

**Build:** The new annealing temperatures were 66°C and 72°C for the GOI and the plasmid respectively. We decided to make four different samples for the thermocycling reaction, trying the annealing temperatures of 66°C and 72°C for both the GOI and the plasmid.

Sample 1. GOI (Tm = 66°C)
Sample 2. GOI (Tm = 72°C)
Sample 3. Plasmid (Tm = 66°C)
Sample 4. Plasmid (Tm = 72°C)

**Test:** We ran the PCR reaction for all 4 samples and analyzed the results using gel electrophoresis on a 1% agarose gel.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/july-18-2022-gel.jpg)

***Figure 9.** 1% agarose gel of the insert and plasmid PCR products. Well 1: DNA ladder, Well 2: Plasmid Tm = 66°C, Well 3: GOI Tm = 66°C, Well 4: GOI Tm = 72°C, Well 5: Plasmid Tm = 72°C*

**Learn:** From the results of this cycle of PCR, we learned that lower annealing temperatures are better suited for the amplification of the GOI and the plasmid. We saw significant DNA laddering and nonspecific bands. We thought that the appearance of nonspecific bands on the PCR gel and the resulting unsuccessful cloning reactions might be due to high GC content of the codon optimized PROS gene and the designed primers as well. Therefore, for our next cycle of DBTL, we decided to perform thermocycling for the GOI and the plasmid at lower annealing temperatures and use GC enhancers for these PCR reactions.

## *Cycle 4: Testing Multiple Lower Annealing Temperatures Along with GC Enhancer*

**Design:** Since we obtained better results by amplifying the GOI and the plasmid at lower annealing temperatures, we decided to try 3 different annealing temperatures along with GC enhancers for the thermocycling reaction. We hoped that using multiple annealing temperatures along with using GC enhancers will increase our chances of improving the thermocycling conditions and get higher quality PCR products for the subsequent cloning step.

**Build:** We made a total of 8 PCR substrate samples, trying annealing temperatures of 55°C and 60°C for the GOI and the plasmid. Moreover, we added GC enhancers to only half of the samples and used the rest non-GC enhanced samples as a control.

Sample 1. GOI (Tm = 66°C)
Sample 2. GOI (Tm = 72°C)
Sample 3. GOI (Tm = 66°C)
Sample 4. GOI (Tm = 72°C)
Sample 5. Plasmid (Tm = 66°C)
Sample 6. Plasmid (Tm = 72°C)
Sample 7. Plasmid (Tm = 66°C)
Sample 8. Plasmid (Tm = 72°C)

**Test:** Each sample was created with identical reagent concentrations and placed into the thermocycler for PCR amplification. The PCR results were analyzed using gel electrophoresis on a 1% agarose gel.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/july-20-2022-gray-gel.jpg)

***Figure 10.** 1% agarose gel of the insert and plasmid PCR products. G + indicates GC enhancer was added, G - indicates GC enhancer absent.*

**Learn:** The results of this cycle of PCR suggest that addition of GC enhancers did not improve the DNA laddering of the PCR products. Referring back to all the cycles of PCR we underwent, we decided that the optimal annealing temperatures for the GOI and the plasmid are 55°C and 62°C respectively as these conditions show bands with the least amount of laddering. We were able to successfully amplify our DNA sequences using these conditions, and obtain pure samples following gel extractions.

## *Cycle 5: Gel Extraction for More Pure and Higher DNA Yield*

**Design:** Even after deciding the best conditions for PCR, we still had some DNA laddering possibly because of non-specific annealing, however, we did get proper amplification of the DNA sequences as indicated by bright bands of the correct size. Therefore, we decided to perform gel extractions to overcome the issue of non-specific bands on the gel and obtain pure DNA samples for cloning.
By loading multiple PCR reactions into one big well on the gel electrophoresis, we hoped to gel extract a significant quantity of pure DNA samples by cutting out just the right sized bands of DNA.

**Build:** Four 50 ug samples of the gene from the PCR reaction were selected. Each one of these samples had mild laddering and a bright band where the DNA of interest would be. These samples were pooled and mixed with gel stain in a 1:5 ratio (gel stain: PCR product). This sample was loaded into 0.7% gel.

**Test:** The band of DNA that contained the gene was cut very closely to prevent excess agar from entering the solution. Furthermore, the cutting process had to occur quickly so that the DNA would not be denatured by the UV imaging system. The sample was washed twice rather than three times to prevent excess loss of DNA. The DNA was suspended in 21 uL of elution buffer to keep DNA concentrations high.

**Learn:** Gel extraction produced a higher yield and concentration when compared to ethanol precipitation. When the gel extraction sample was tested on gel electrophoresis, only one band of DNA appeared brightly. All future DNA samples that were used for the LIC went through gel extraction.

## *Cycle 6: Ligation Independent Cloning of the GOI and the Plasmid Substrates*

**Design:** After obtaining pure DNA samples by gel extraction of the PCR products, we treated both the GOI and the plasmid for the T4 polymerase 3’ chewback step. We used a ratio of approximately 3:1 (Insert:Plasmid), and about 38 ng of the insert vs 14 ng of the plasmid for the annealing reaction and transformed them into *E. coli* NovaBlue cells.

**Build:** Since our first LIC attempt and transformations into NovaBlue cells were unsuccessful, we reattempted LIC at various conditions and DNA concentrations. We consistently failed with an insert:plasmid ratio of 3:1 and increasing their concentrations as well, therefore, we decided to increase the final concentrations of the insert and the plasmid to 20 ng/uL in the 20 uL LIC reaction mix. We used 400 ng of both the insert and the plasmid with a ratio of 1:1 (Insert : Plasmid) for this round of LIC reaction.

**Test:** We mixed 5 uL of both the insert and the plasmid LIC products, vortexed and let it sit at room temperature for 30 min for the annealing to occur. We transformed the LIC mixture into DH5alpha and plated the cells after 1 hour of incubation.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-21-lic-3.png)

***Figure 11.*** Transformation plates of DH5 alpha

No colonies were observed on the negative control plate (linearized plasmid). However, we observed some colonies on the experimental plate. We picked 2 colonies (circled using a blue marker) to perform a colony PCR analysis.

We picked what seemed like bacterial colonies from the experimental plate and inoculated them overnight to perform a colony PCR analysis.

![](https://static.igem.wiki/teams/4235/wiki/engineering-success/aug-3-2022.jpg)

***Figure 12.** 0.8% agarose gel of the colony PCR products*

| Well Set-Up | | | | | | | || Well # | Well Setup |
| Well 1 | DNA ladder |
| Well 2 | Colony 1 (Original Primers, Tm = 62°C ) |
| Well 3 | Colony 1 (Control Primers, Tm = 55°C ) |
| Well 4 | Colony 2 (Original Primers, Tm = 65°C ) |
| Well 5 | Colony 2 (Control Primers, Tm = 55°C ) |
| Well 6 | Control Plasmid |

***Table 4.** Colony PCR gel well setup.*

**Learn:** According to Figure 12, Colony 1 seemed to have our recombinant vector, as we could see bands of approximately 2 kB size, which matched with the weight of our insert ~ 2kB. However, we were unable to grow colonies from the successful DH5alpha transformation plate.
Because of time constraints, we decided to divert all of our resources towards developing our bacterial expression systems.

Results from the colony PCR indicated presence of the recombinant vector in Colony 1, however, we decided that this alone was not a definitive proof of successful cloning, as we had to discontinue our lab experiments for SF9. We were successful in optimizing the PCR conditions for our GC rich insert and plasmid sequences, and possibly also optimized conditions for the Ligation Independent Cloning.

*## Conclusion*

We went through multiple cycles of design and testing to optimize the conditions for PCR amplification of our highly GC rich DNA sequences and possibly also the LIC reaction.

However, we were not able to continue our lab efforts past the colony PCR, which by itself is not sufficient proof of successful cloning. We discontinued the progress for the sake of focusing on performing successful expression in *E. coli* cell lines, which we achieved a month and a half later. However, by showcasing our work with SF9, we hope that future teams, given adequate time and resources, will be able to further optimize the wet lab experiments in order to successfully achieve expression from SF9 cells. This would be the first step in industrializing the process and bringing another option except for expression for creating an injectable protein S therapy.

Considering we have adequate time to continue our SF9 experiments, our next steps would be to purify enough of our recombinant plasmid by mini-prep in order to be able to sequence it. If confirmed by sequencing, further steps would include transformation into *E. coli* DH10Bac and following the Baculovirus expression system protocols.

![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
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---

### Discover

[![](https://static.igem.wiki/teams/4235/wiki/footers/footer-7.jpg)](https://2022.igem.wiki/stony-brook/parts)

## Parts

Genetic parts which we utilized in our system.

[Learn More](https://2022.igem.wiki/stony-brook/parts)

[![](https://static.igem.wiki/teams/4235/wiki/footers/footer-4.png)](https://2022.igem.wiki/stony-brook/human-practices)

## Human Practices

An analysis of how our work affects the world,
 and how the world affects our work.

[Learn More](https://2022.igem.wiki/stony-brook/human-practices)

{% endblock %}

# Wet Lab Notebook

Here we provide a list of all the experiments conducted as a part of our wet lab and the protocols we referred to. Our protocols were compiled by conducting literature searches and other sources. We would like to express our sincere gratitude to Douglass Marr and Andrew Sillato for sharing their lab protocols and resources, and for their unconditional support throughout our project.

---

### Complete Lab Protocols

Please click the link below to access our detailed lab notebook!

[E. coli Wet Lab Notebook](https://docs.google.com/document/d/1fDVJFbCkUY8ycaGYMtppeAhLANUYO05aZW65nvQInns/edit?usp=sharing)

Below is a list of our protocols

* [PCR](https://drive.google.com/file/d/17s-ToCzrSJG16BNIppY8mmwilIv2nAi3/view?usp=sharing)
* [Ligase Independent Cloning (LIC)](https://docs.google.com/document/d/1W7TM6xVg_KQcajaUfkrDh_1Etu8ztQ4twf0eQ1tK2so/edit?usp=sharing)
* [QIAprep Spin MiniPrep Kit protocol](https://drive.google.com/file/d/1hvpX_U-XSDupEMtJ4hpdoz5nnkpJVLqZ/view?usp=sharing)
* [SDS-PAGE](https://docs.google.com/document/d/1BRKhvqI0b-BI4YVls0SVgXHsxJg0aIXzG3apavoA9to/edit?usp=sharing)
* [Bacterial Transformation](https://drive.google.com/file/d/1BlC3pkNfR0EA7HuqTh1K_Dse0olUHRyO/view?usp=sharing)
* [Western Blot](https://drive.google.com/file/d/1CHbIgl5hA5KT2hcOJzWqRuZuRMDOYZ7d/view?usp=sharing)

A timeline of our experiments can be found below.

---

### Timeline

## *Gene and Vector Amplification (July 6 - July 20)*

The gene of interest PROS was amplified using PCR. We ran a gel to check if the PCR was successful. Cultures of DH5-alpha E. coli cells containing 2Bc-T plasmid were grown overnight and miniprepped. Concentrations of the gene and plasmid were recorded. The miniprepped 2Bc-T plasmid was then amplified by PCR and we ran a gel to check if the PCR was successful. This process was repeated and troubleshooted multiple times.

## *Restriction Digest of Vector (July 25)*

The 2Bc-T plasmid was linearized using the HpaI restriction enzyme and confirmed by a gel.

## *LIC Reaction for Insert (July 26)*

A LIC reaction was performed for our PROS insert and concentration was confirmed.

## *Vector Growth and Purification (July 26 - July 27)*

Cultures of DH5-alpha E. coli cells containing 2Bc-T plasmid were grown overnight and miniprepped. Concentrations of the plasmid were recorded.

## *Restriction Digest of Vector (July 27 - August 2)*

The 2Bc-T plasmid was linearized using the HpaI restriction enzyme and confirmed by a gel. This process was repeated and troubleshooted to ensure linearization took place.

## *Extraction of Linearized Vector (August 2 - August 3)*

The linearized 2Bc-T vector was extracted from the gel using gel extraction and concentration was confirmed. A LIC reaction was performed for the gel extracted and linearized vector.

## *Annealing of Vector and Insert (August 4)*

The linearized 2Bc-T vector and PROS gene of interest were annealed to produce a recombinant vector.

## *Transformation into DH5-alpha E. coli Cells (August 4 - August 14)*

The recombinant vector and a positive control vector (only plasmid) were transformed into DH5-alpha E. coli cells to determine if the recombinant vector was made successfully. This was initially unsuccessful so all of the previous processes entailing making the recombinant plasmid were repeated. On the second try, there were colonies present, indicating successful transformation.

## *Transformed Cells’ Growth & Purification (August 15 - August 17)*

The colonies from the transformation were grown overnight and miniprepped. The concentration was measured.

## *Digestion of Recombinant Vector (August 17)*

We performed a restriction digest of the recombinant vector with Xho1 restriction enzyme. We ran a DNA gel with the product to ensure annealing took place, which yielded positive results and indication that the recombinant vector was present in the samples.

## *Amplification of Recombinant Vector (August 18)*

A PCR of the miniprepped recombinant vector was performed. We ran a DNA gel to ensure that the samples contained the recombinant vector, which was successful.

## *Transformation of BL21 and Origami E. coli Cells (August 19)*

The recombinant vector was transformed into BL21 and Origami E. coli cells for protein expression. The transformations were successful.

## *Growth of Transformed Cells (August 23 - August 24)*

Cultures of Origami and BL21 E. coli cells containing the recombinant vector were grown overnight.

## *SDS-PAGE and Western Blot (August 27 - September 3)*

Analytical techniques such as running an SDS-PAGE gel and Western Blot were performed to ensure that Protein S was made. The HRP anti-His 6 antibody successfully detected the Protein S target band at about 80 kD.

---

### SF9 Wet Lab Notebook and Protocols

Please click the link below to access our detailed lab notebook!

[SF9 Wet Lab Notebook](https://docs.google.com/document/d/1Jqz-252bXJ__4mEx_28zRImMixBBkVyezek2SamE6uY/edit?usp=sharing)

Below is a list of our protocols

* [PCR Protocol Optimized for the PROS Gene and Colony PCR Protocol](https://docs.google.com/document/d/18ten1U42J9OOK96HMe-MsvJYR5WpAQwmFcPfw2vM7lk/edit?usp=sharing)
* [Touchdown PCR](https://docs.google.com/document/d/1GrwY2DPG2e2lp8S8SfyHWJ_mF5Spesdr0f25i_bXQTo/edit?usp=sharing)
* [Ligase Independent Cloning (LIC)](https://docs.google.com/document/d/1SPFJUXMUZ3NBRZ9AIhgKj-fQEqwbBFxEibFZ9wjfgCE/edit?usp=sharing)
* [Restriction Digest](https://docs.google.com/document/d/197jpmHXAvoMZACZgu9QX9_IvwZ_Q1SjDaamOSrIAOPc/edit?usp=sharing)
* [6XHis Lyticase Purification Protocol](https://docs.google.com/document/d/1jRSBprd9eIxaGDg2bRE3VrE8N0a_tMv_/edit?usp=sharing&ouid=117552519386159979138&rtpof=true&sd=true)
* [Competent Cell Preparation](https://docs.google.com/document/d/1v57HHmpH5Jq_woKfNLCu_sjw-WPJ5C_k/edit?usp=sharing&ouid=117552519386159979138&rtpof=true&sd=true)
* [*E. coli* Transformation Protocol](https://docs.google.com/document/d/1xnSVDRsn0ld4LLw7qbTuyFORAJ6lTnRdte1feI9mPEc/edit?usp=sharing)
* [Thermo Scientific Gel Extraction Kit Protocol](https://drive.google.com/file/d/10Uj8TTtRNRPUXBt_ZAaCtKmMF1jy0eAM/view?usp=sharing)
* [SF9 Cells Passaging Protocol](https://docs.google.com/document/d/1mDM2x-lRr1oAonCoJQPv8dLRO_X7Xp5zaHm_wQH7PLI/edit?usp=sharing)
* [Primer Design Guidelines](https://docs.google.com/document/d/1qxouLDb0c-hsAhEzKXovMr9MN3a0R6oLC-qHA1GaUu8/edit?usp=sharing)
* [Monarch PCR and DNA Cleanup Kit](https://docs.google.com/document/d/1p_wkjtfyCIWEZ1zPu-JQylu97RMZJTwJTLiyjYbHbwA/edit?usp=sharing)
* [SF9 Transfection Protocol](https://docs.google.com/document/d/1H8vsAi819bEHKiBg19jt5oI2lw9Ly9xGAjXyI5_a35g/edit?usp=sharing)
* [Ethanol Precipitation for Concentration of DNA Samples](https://docs.google.com/document/d/1VmqHSf6FqHm08SOao5H_i2wVojcJl55YK773IusjF1E/edit?usp=sharing)
* [Bac-To-Bac Baculovirus Expression Systems](https://drive.google.com/file/d/1Ehl1mctfgjaOO-Ji_dtgXGz9k5NiWnp0/view?usp=sharing)

A timeline of our experiments can be found below.

---

### Timeline

## *PCR of new primers onto the PROS gene and the YMBacII vector (July 1-6)*

We ordered the GOI with other primers attached to its ends. We need to use different primers, however. The new primers are compatible with YMBac II plasmid, into which the GOI (with new, compatible primers), will be PCR-ed into. We used Protocol for Q5® High-Fidelity 2X Master Mix. We ran a DNA gel with both products of the PCR reaction (modified YmBac II and modified PROS) to see if they have been amplified and have correct weight. They had the correct weight and good amplification, but there was significant laddering for the gene, so we had to optimize the conditions. We then performed PCR product clean up and nanodrop to determine the concentration of the gene and the vector. We made calculations to find the volume of cleaned up PCR product needed for LIC.

## *Making Ampicillin LB agar plates to grow the transformants on, and plating Nova Blue *E. coli* with the plasmid and gene cloned into them (July 7-10)*

We saw no growth on any of the plates, so we decided to perform a positive control using intact YMBcII.

*## Running a positive control using intact YMBacII plasmid: transforming Nova Blue with intact plasmid and plating them (July 11)*

Again, we saw no growth which suggested that the mistake is with either our gene and vector or with the technique of transforming or the cells.

*## Re-adding primers to the gene and to the vector by PCR (July 12)*

We ran a gel of the results, and the bands were extremely faint, so we decided to troubleshoot the amplification and optimize the PCR.

*## Optimization of PCR for both vector and gene (July 13 - July 26)*

We recalculated annealing temperature with corrected values, and tried different annealing conditions such as 72°C, 66°C, 62 °C, 55°C, increased annealing times, and using GC enhancers.
At times our PCR reactions failed completely as proven by nanodrop, which we assumed was either human error or machine error. This could also be due to the fact that our primers were highly GC rich and could not be redesigned because of the vector we had to use. The PCR reaction was very difficult to get to work, but at last we found the perfect conditions which allowed reproducible good amplification, and minimized laddering. However, we still had to gel extract since the laddering was still significant. We managed to gel extract at very high concentration and purity (vector concentration 47.3 ng/uL and purity 1.83 where 1.80 is the ideal purity, as well as gene concentration of 56.8 ng/uL with 1.87 purity), which is more than enough for the purposes of LIC, especially that we had about 20 ul of the gel-extracted vector.

## *LIC (cloning the insert into the vector) and transformation of the LIC assembled vector into Nova Blue *E. coli* (July 26-August 3)*

The first attempt was unsuccessful, so we tried it again with new conditions (lowered the time of heat shock and ice incubation). Again, no colonies formed for the second time either. For the third time, we did achieve two successful colonies. We cultured them, but the cells did not grow in culture. We tried a colony PCR to verify whether the cells had the correctly cloned insert + vector, and ran a gel. The weight of the vector that was supposed to be achieved was clearly visible on the colony PCR gel. This partially supports the claim that we achieved engineering success in creating a construct optimized for the furthering of SF9 protein S expression. To have better evidence, the next step would be to purify enough of this vector by mini-prep to be able to sequence it. Once we would sequence it, we would be sure about the success of the transformation. Further steps could include checking if we need to design primers for sequencing, and making a new 5 ml LB colony so we can sequence, since we would need to get 300-400 ng of vector to sequence. However, at this point in the experiment we did not have the time or resources to continue with SF9 and we decided to continue solely with *E. coli* expression.

![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)

---

### Discover

[![](https://static.igem.wiki/teams/4235/wiki/footers/footer-5.png)](https://2022.igem.wiki/stony-brook/model)

## Model

Our dynamic mathematical models
and computer simulations.

[Learn More](https://2022.igem.wiki/stony-brook/model)

[![](https://static.igem.wiki/teams/4235/wiki/footers/footer-6.png)](https://2022.igem.wiki/stony-brook/entrepreneurship)

## Entrepreneurship

Our efforts to build a business case
and commercialize our product.

[Learn More](https://2022.igem.wiki/stony-brook/entrepreneurship)

{% endblock %}

# Results

![](https://static.igem.wiki/teams/4235/wiki/b7491679-a831-4ad8-98b4-e7393099f6fa.png)The results of our construct designs and corresponding ligations, transformations, and expression tests are highlighted on this page. Our overall goal to was to introduce foreign protein S gene into the plasmids [pFastBac-YMBac-II](http://parts.igem.org/Part%3ABBa_K4235002) and [pET His6 (2Bc-t)](http://parts.igem.org/Part%3ABBa_K4235016) and transform into *E. coli* DH10Bac cells containing the Bacmid for generating recombinant viral particles in order to achieve expression in SF9 cells using the Bac to Bac baculovirus expression system, and transforming into *E. coli* cell strains: *E. coli* Bl21, Origami B (DE3), Origami 2 (DE3) and Origami 2 (DE3) pLysS. Conducting parallel experiments with eukaryotic and prokaryotic expression systems enabled us to understand the feasibility and ability of these expression systems to produce recombinant human protein S. We were also able to generate data that helped us develop an implementation plan to scale up protein S production. In the future, this will help our project on an industrial level, as we aim to create an injectable therapeutic, PROS, for people with protein S deficiency. We were not able to achieve expression from the baculovirus expression system because of time constraints, but we were successful in expressing protein S in Origami B (DE3) cells.

---

### Prokaryotic Expression (*E. coli*)

## *Verification of Synthesized Insert and Linearized Vector*

We obtained our gene of insert through synthesis from IDT. We amplified the insert, which was to be cloned into our vector, using primers designed for the Ligation Independent Cloning reaction. The figure below shows successful amplification of the synthesized gene, containing the overhangs designed for LIC.

![](https://static.igem.wiki/teams/4235/wiki/results/insert-pcr.jpeg)

***Figure 1.*** PCR amplification of the insert using LIC primers. Well 3 contains the NEB 1kb plus DNA ladder and well 4 is the gene of interest (2kb band). This indicates that the insert is successfully amplified using LIC primers.

We received our [expression vector](http://parts.igem.org/Part%3ABBa_K4235016) as a generous donation from the Glynn lab at Stony Brook University. We grew overnight cultures of *E. coli* DH5-alpha cells containing our vector. In total we inoculated 5 different 5 mL bacterial cultures labeled Samples J, K, L, M and N and cultured them overnight at 37°C and 220 rpm. The figure below shows the gel electrophoresis result of PCR amplification of the mini prepped vector using LIC primers.

![](https://static.igem.wiki/teams/4235/wiki/results/miniprep-gel-results.jpeg)

***Figure 2:** Gel electrophoresis of the mini prepped plasmid BBa\_K4235016. DH5alpha containing the plasmid were inoculated overnight: 4 culture tubes labeled: K,L,M and N, each with 5 mL LB and 2.5 uL ampicillin (50 ug/uL working concentration). Sample J was obtained from an older miniprep.*

## *Construction of the Recombinant Vector*

The insert was optimized for the annealing reaction by generating specific overhangs for the LIC reaction by PCR amplification. The vector has a LIC site which is acted upon by the restriction enzyme Hpal, to linearize the vector prior to the LIC reaction. Following are the primer sequences used to generate the complementary overhangs for the annealing reaction;

**Forward primer:** TTTAAGAAGGAGATATAGTTCATGCGCGTACTTGGCGGACGC

**Reverse primer:** GGATTGGAAGTAGAGGTTCTCAGAGTTCTTAGTTTTTTTCCAAACTG

After a lot of [troubleshooting](https://2022.igem.wiki/stony-brook/engineering), we determined the optimal insert and vector concentration ratios for the ligation independent cloning (LIC) reaction. For our annealing reaction, we conducted two parallel LIC reactions:

||  |  |
| --- | --- |
| Reaction | Concentrations |
||  |  |
| --- | --- |
| Reaction 1 | 2 uL of insert LIC product + 10 uL of vector LIC product + 8 uL Molecular grade H2O |
| Reaction 2 | 5 uL of insert LIC product + 10 uL of vector LIC product + 5 uL Molecular grade H2O |

***Table 1.*** Concentrations of the parallel LIC reactions. The concentrations of the insert and vector after the LIC reaction were 5.55 ng/uL and 68.35 ng/uL, respectively.

## *Transformation of the Recombinant Vector Into DH5-alpha*

Once the recombinant vector was successfully annealed, the vector had to be transformed into DH5-alpha *E. coli* cells as an experimental transformation before transformation into cells that would be used for protein expression. Two different reactions were set up in order to increase the chances of transformation success. The difference between the two reactions was the volume of the gene of interest used for annealing. Both reaction samples sat on ice for 30 minutes, were heat shocked at 42°C, incubated at 37°C for one hour, centrifuged, and resuspended in remaining LB broth. Two different ampicillin agar plates were set up, in which 200 uL of a reaction was plated. After overnight incubation at 37°C, both plates showed successful growth of colonies, indicating that the transformation of the recombinant vector in DH5-alpha *E. coli* cells was successful.

![](https://static.igem.wiki/teams/4235/wiki/results/lic-transformants.jpeg)

***Figure 3.** Transformation plates of DH5alpha. We transformed our annealed construct after the LIC reaction into Dh5alpha. Colonies growing on Ampicillin plates confirm the presence of recombinant vector.*

## *Verifying Gene Insertion Into the Vector*

We selected three colonies from each of the post-LIC transformed DH5-alpha plates.

Reaction 1: Samples 1D, 1E and 1F.

Reaction 2: Samples 2D, 2E and 2F.

We performed a restriction digest analysis using enzymes xho1 and BamH1. The figure below shows the gel electrophoresis result of the restriction digest.

![](https://static.igem.wiki/teams/4235/wiki/results/restriction-digest.jpeg)

***Figure 4.*** Gel electrophoresis results from the restriction digest on the recombinant vector. There colonies were picked from each Dh5alpha transformants. Reaction 1 and reaction 2 were inoculated overnight. We performed a restriction digest analysis on the mini prepped recombinant vector using enzymes xho1 and Bamh1. Samples from Reaction 1 are labeled as 1D, 1E, 1F and samples from reaction 2 are labeled as 2D, 2E, 2F.

From the results we concluded that only one of the two restriction enzymes used for the digest functioned and linearized the vector. Since the bands of samples 1E and 1F were around 7-8 Kb, we concluded that these samples contain our recombinant plasmid. Well 1 contained 4 uL 1 Kb plus ladder, Well 2 contained the plasmid before restriction digest, Wells 3-8 contained samples 1E-2F.

To verify that samples 1E and 1F contained our recombinant plasmid, we used T7 forward and reverse primers to run a PCR amplification over the multiple cloning site. The amplicon resulting in a band around 2 Kb would indicate successful cloning of our insert into the vector. The figure below shows the results of PCR amplification.

![](https://static.igem.wiki/teams/4235/wiki/results/recombinant-vector-pcr.jpeg)

***Figure 5.** PCR amplification of the recombinant vector for insert verification*

As evident from figure 5, sample 1E generated an amplicon of size between 2-3 Kb and contained our recombinant vector. This indicates that the Protein S gene was successfully cloned into the pET His6 LIC cloning vector (2Bc-T).

## *Sequencing of the Recombinant Vector*

In addition to utilizing the gel to confirm the recombinant vector, the following image displays the sequencing result that was performed using the T7 forward primer. The NCBI BLAST tool was used for pairwise alignment of the sequenced DNA vs protein S insert sequence. The percent match of 97.37% indicates that the gene was successfully inserted into the vector.

![](https://static.igem.wiki/teams/4235/wiki/results/seq-t7-f.png)

![](https://static.igem.wiki/teams/4235/wiki/results/result1.png)

***Figure 6.*** NCBI BLAST pairwise alignment results.

The following image displays the sequencing result of the recombinant vector using the T7 reverse primer. The percent match of 95.72% indicates that the gene was successfully inserted into the vector.

![](https://static.igem.wiki/teams/4235/wiki/results/seq-t7-rev.png)

![](https://static.igem.wiki/teams/4235/wiki/results/result2.png)

***Figure 7.** NCBI BLAST pairwise alignment results.*

DNA sequencing of our recombinant vector using the T7 forward and reverse primers confirm successful cloning of our insert into the Multiple Cloning Site of the vector using ligation independent cloning (LIC).

## *Transformation into *E. coli* Cell Strains for Prokaryotic Expression*

For selecting the optimal expression strains, we transformed our recombinant vector into four different *E. coli* strains: Bl21, Origami B (DE3), Origami 2 (DE3) pLysS and Origami 2 (DE3). Recombinant vector was transformed into these four cell strains which were then inoculated in 1 mL of LB broth and placed in an incubator shaker at 37°C, 220 rpm for 1 hour. 150 uL from each cell culture were plated and the results are as follows:

![](https://static.igem.wiki/teams/4235/wiki/results/bl21-transformant.jpeg)

***Figure 8.** BL21 transformation plate
(LB agar with ampicillin)*

![](https://static.igem.wiki/teams/4235/wiki/results/origami-transformant.jpeg)

***Figure 9.** Origami B transformation plate
(LB agar with ampicillin)*

![](https://static.igem.wiki/teams/4235/wiki/results/origami-s-transformation.png)

***Figure 10.** Origami S (Origami 2 (DE3) pLysS) transformation plate
(LB agar with ampicillin)*

![](https://static.igem.wiki/teams/4235/wiki/results/origami-x-transformant.png)

***Figure 11.** Origami X (2) (Origami 2 (DE3)) transformation plate
(LB agar with ampicillin)*

We observed recombinant colonies on the transformation plates of Bl21 and Origami B cell strains. No colonies were observed for Origami S and Origami X (2) transformation plates. Colonies were selected from Bl21 and Origami B transformation plates for conducting recombinant expression tests.

## *Detection of Protein Expression From the Selected Bacterial Strains*

We conducted two expression tests to confirm production of recombinant protein S in the transformed cell strains: *E coli*. BL21 and origami B cells. For the first expression test, we picked colonies from the transformation plates containing our recombinant vector, cultured them in 5 mL LB and ampicillin overnight at 37°C, and used 125 mL of the overnight culture to dilute it into a 25 mL culture. Both the 25 mL cultures of Bl21 and origami B cells were grown to OD600 at 37°C and induced using 1 mM IPTG. A pre-induction sample was obtained from both cultures before induction. After induction, the cultures were grown for 4 hours at at 37°C and 220 rpm, and a post-induction sample was obtained every hour post-induction for 4 hours. All the expression samples were spun down at 3000 rpm for 5 min to harvest the cells, the supernatant was discarded, and the pellet was stored for conducting the expression tests. Below are the results of the SDS-PAGE and the western blot of our expression test:

![](https://static.igem.wiki/teams/4235/wiki/results/result3.png)

***Figure 12.** SDS-PAGE of the BL21 and Origami B expression samples.*

||  |  |
| --- | --- |
| Well # | Sample |
||  |  |
| --- | --- |
| Well 1 | Ladder |
| Well 2 | Origami B - Pre-Induction |
| Well 3 | Origami B - 1 hr Post-Induction |
| Well 4 | Origami B - 2 hr Post-Induction |
| Well 5 | Origami B - 3 hr Post-Induction |
| Well 6 | Origami B - 4 hr Post-Induction |
| Well 7 | BL21 - 1 hr Post-Induction |
| Well 8 | BL21 - 2 hr Post-Induction |
| Well 9 | BL21 - 3 hr Post-Induction |
| Well 10 | BL21 - 4 hr Post-Induction |

***Table 2.** SDS-PAGE well setup*

![](https://static.igem.wiki/teams/4235/wiki/results/result4.png)

***Figure 13.** Western blot displaying the first expression test result.*

||  |  |
| --- | --- |
| Well # | Sample |
||  |  |
| --- | --- |
| Well 1 | Ladder |
| Well 2 | Origami B - Pre-Induction |
| Well 3 | Origami B - 1 hr |
| Well 4 | Origami B - 2 hr Post-Induction |
| Well 5 | Origami B - 3 hr Post-Induction |
| Well 6 | Origami B - 4 hr Post-Induction |
| Well 7 | BL21 - 1 hr Post-Induction |
| Well 8 | BL21 - 2 hr Post-Induction |
| Well 9 | BL21 - 3 hr Post-Induction |
| Well 10 | BL21 - 4 hr Post-Induction |
| Well 11 | Ladder |

***Table 3.** Western blot well setup.*

However, the first expression test was not entirely conclusive. There was no expression from BL21 as indicated by the western blot results. Furthermore, the bands produced for Origami B expression samples were of incorrect size. Protein S is about 70 kD and the bands on the Western blot were around 30 kD. From the first expression test, it was concluded that BL21 is unable to express protein S as it might not be able to form the necessary glycosylations and disulfide bonds, which would result in the protein being insoluble and aggregating in the cells. The incorrect size bands of the Origami B protein sample indicates that the protein is being degraded after expression, which can be as a result of expressing at 37°C and inducing with 1 mM IPTG.

## *Optimizing Protein Expression*

After consultation with advisors, we decided to perform another expression test on origami B cells. To optimize the expression, the origami cells were induced at 37°C and the temperature was gradually reduced to 23°C and induced using 0.5 mM, instead of 1 mM like in the first expression test. Two origami samples at different cell concentrations were also induced in parallel. Moreover, the induction samples were also sonicated (to lyse the cells) for 1 minute and protease inhibitors were added before performing the western blot.

Sample Ori 2 was induced at OD = 0.4 and sample Ori 3 was induced at OD = 1.1. The one pre-induction sample was collected from both Ori 2 and Ori 3 before inducing with 0.5 mM IPTG and 4 post-induction samples at an increment of 1 hour. Sample Ori 2 was expressed for about 16 hours and ori 3 for about 12 hours to collect overnight induction samples.

The following image shows western blot results for the second expression test:

![](https://static.igem.wiki/teams/4235/wiki/results/final-western-blot.jpg)

***Figure 14.** Western blot of the second expression test.*

Well setup is as follows:

1. Pre-induction Sample (Ori 2)
2. One hour post-induction (Ori 2)
3. Two hour post-induction (Ori 2)
4. Three hour post-induction (Ori 2)
5. Four hour post-induction (Ori 2)
6. Overnight induction (Ori 2)
7. Empty well (loading dye)
8. Pre-induction Sample (Ori 3)
9. One hour post-induction (Ori 3)
10. Two hour post-induction (Ori 3)
11. Three hour post-induction (Ori 3)
12. Four hour post-induction (Ori 3)
13. Overnight induction (Ori 3)

As evident from Figure 14, the HRP anti-His 6 antibody successfully detected protein S target band (full length, about 70 kD) by Western blot. The level of expression can be seen increasing with time for sample Ori 3. As expected, the ability of origami B cells to make disulfide bonds and ensure proper folding, greatly enhances protein stability and prevents degradation as opposed to the BL21 strain. Moreover, the multiple bands visible from about 30-50 kD indicates that the protein is still being degraded from the N-terminal, since we have detection of the 6XHis-tags from the C-terminal. Protein S has multiple N-linked Glycans added as a part of its post-translational modification, which might have protective functions for the EGF domains and the disulfide bonds. The inability of our prokaryotic expression system to introduce glycosylations might be responsible for the degradation of the protein. We believe that expressing glycosyltransferases in both bacterial and yeast cell lines is a promising avenue for future research and a first step in increasing the resemblance of the in-cell produced protein S to human protein S.

---

### Eukaryotic Expression (SF9)

In order to develop SF9 baculovirus expression system for recombinant protein S production, we planned on constructing a recombinant transfer vector [pFastBac-YMBac-II](http://parts.igem.org/Part%3ABBa_K4235002), which would involve cloning our [insert](http://parts.igem.org/Part%3ABBa_K4235000) into the MCS of this vector, which is within the transposon segment (mini-attTn7). We planned on inserting our gene of interest into the MCS of the plasmid such that we have an RsrII restriction site just upstream of our insert and a TEV protease site, a twin-strep tag and 6X His-tag downstream of our insert. The sequences of our plasmid and the gene of interest used to design primers for the LIC reaction were highly GC rich, and we had to undergo several cycles of testing and improvement to optimize PCR conditions for our gene and the plasmid. After optimizing the conditions for PCR, we tested multiple conditions to optimize the Insert to vector ratio and concentrations for the LIC reaction.

## *Amplification of the Gene of Interest and the Plasmid*

Our initial step was to amplify our GOI and plasmid using the primers we designed for generating 5’ overhangs after the T4 polymerase check back step for the Ligation Independent Cloning reaction. For this PCR we used an annealing temperature of 55°C.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-15-pcr-1.png)

***Figure 15.*** 1% agarose gel of the insert and plasmid PCR products. Well 3: DNA ladder, Well 4: Gene of interest PCR product, Well 5: Plasmid PCR product

As evident from figure 15, the gene of interest had been successfully amplified, however, there was significant laddering for the plasmid, which can be a result of non-specific annealing of the primers. Therefore, we decided to further optimize our PCR conditions.

## *Testing Multiple Annealing Temperatures for the Gene of Interest and the Plasmid*

We changed the PCR conditions for the plasmid by increasing the extension time from 2 to 2.5 minutes and the final extension time from 2.5 to 5 minutes. We redid the PCR on our plasmid at 2 different annealing temperatures: 55°C and 62°C.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-16-pcr-2.png)

***Figure 16.** 1% agarose gel of the plasmid PCR products. Well 1: DNA ladder, Well 2: Plasmid Tm = 55°C Well 3: Plasmid Tm = 62°C*

We decided to further optimize the PCR conditions as we could still observe some laddering on the gel, which might have been a result of the non-specific annealing of the primers.

For the next cycle of the PCR, we tried annealing temperatures of 66°C and 72°C for both the gene of interest and the plasmid.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-17-pcr-3.png)

***Figure 17.** 1% agarose gel of the insert and plasmid PCR products. Well 1: DNA ladder, Well 2: Plasmid Tm = 66°C, Well 3: GOI Tm = 66°C, Well 4: GOI Tm = 72°C, Well 5: Plasmid Tm = 72°C*

The results of this PCR cycle indicated that lower annealing temperatures are preferable for both the GOI and the plasmid. Moreover, we decided to use GC enhancers for setting up the PCR reaction samples for both the GOI and the plasmid, with the hope of reducing non-specific annealing and improving the quality of the PCR amplified products.

For the next cycle of PCR, we decided to run a PCR on the GOI at annealing temperatures of *55°*C and *60°*C and test the PCR products of both, with and without adding the GC enhancers. Similarly, we decided to use annealing temperatures of *55°*C and *62°*C for the plasmid and test the PCR products of both, with and without adding the GC enhancers.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-18-pcr-4.png)

***Figure 18.** 1% agarose gel of the insert and plasmid PCR products. G + indicates GC enhancer was added, G - indicates GC enhancer absent.*

The results of this PCR cycle indicate that the optimal condition for the plasmid’s PCR reaction is an annealing temperature of 62°C and no GC enhancer present. All the bands for the GOI were very faint, which we concluded was because of the low concentration of DNA used for the PCR.

We re-did PCR on the GOI using an annealing temperature of 55°C, which according to our first PCR cycle, showed the best amplification for the GOI.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-19-pcr-5.png)

***Figure 19.** 1% agarose gel of the insert and plasmid PCR products. Well 1: DNA ladder, Well 2-5: GOI at Tm = 55°C (50 uL sample per well). Well 6: DNA ladder*

Despite trying a range of annealing temperatures, extension and final extension times during PCR and addition of GC enhancers, we were not able to significantly reduce the laddering effect seen on the gels. Therefore, we decided to use the best PCR conditions for the GOI and the plasmid, that we derived through many cycles of testing and learning, and performed gel extractions to obtain pure samples that could be used for the Ligation Independent Cloning reaction.

---

### Optimizing Conditions for the Ligation Independent Cloning Reaction

We tested different ratios and concentrations of the insert and the vector for the LIC reaction. For the LIC reaction, we followed the protocol used by Douglas Marr, from Dr. Michael Airola’s lab at Stony Brook University.

---

## *LIC Attempt 1*

Using the following calculator, we calculated the amount of insert and vector DNA we needed to use for the LIC reaction.

[Ligase Independent Cloning (LIC)](https://www.helmholtz-muenchen.de/fileadmin/PEPF/Protocols/LIC-cloning.pdf)

We used a ratio of approximately 3:1 (Insert:Vector), and about 38 ng of the insert vs 14 ng of the vector.

The protocol for this LIC reaction can be found below:

[LIC Cloning (Douglass Marr, Michael Airola)](https://docs.google.com/document/d/1eIYSVrlToo8UaMpONz1CAcDCaaf0HNU1ouOvA-q8TKY/edit?usp=sharing)

We mixed 2 uL of the vector LIC product and 2 uL of the insert LIC product, vortexed it and let it sit at room temperature for 10 minutes before transforming into *E. coli* NovaBlue cells and plating them.

No colonies were observed on the experimental plate, but several colonies on the positive control plate (just the intact vector pFastBac-YMBacII).

## *LIC Attempt 2*

For the next round of the LIC reaction, we decided to increase the concentration of the insert and the vector used for annealing. We used a ratio of approximately 3:1 (Insert : Vector), and about 142 ng of the insert vs 47 ng of the vector. Moreover, we used *E. coli* DH5alpha for transformation. We used 5 uL of the insert LIC product + 5 uL of the vector LIC product and diluted it using 10 uL of ddH2O making a total volume of 20 uL for transformation.
We used the linearized plasmid pFastBac as a negative control.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-20-lic-2.png)

***Figure 20.** negative control and experimental plates of DH5alpha transformants*

No colonies were observed on either the experimental or negative control plates.

## *LIC Attempt 3*

After consulting with our advisors, Douglass Marr and Andrew Sillato, we decided to increase the final concentrations of the insert and vector to 20 ng/uL in the 20 uL LIC reaction mix. Therefore we used 400 ng of both the insert and the vector for this round of LIC reaction.
We used a ratio of 1:1 (Insert : Vector), and about 400 ng of both the insert and the vector. We mixed 5 uL of both the insert and the vector LIC products, vortexed and let it sit at room temperature for 30 min for the annealing to occur. We transformed the LIC mixture into DH5alpha and plated the cells after 1 hour of incubation.

![](https://static.igem.wiki/teams/4235/wiki/results/fig-21-lic-3.png)

***Figure 21.** negative control and experimental plates of DH5alpha transformants*

As expected, no colonies were observed on the negative control plate (linearized plasmid pFastBac). However, we observed some colonies on the experimental plate. We picked 2 colonies (circled using a blue marker) to perform a colony PCR analysis.

## Colony PCR Analysis

![](https://static.igem.wiki/teams/4235/wiki/results/fig-22-colony-pcr.png)

***Figure 22.*** 0.8% agarose gel of the colony PCR products.

||  |  |
| --- | --- |
| Well # | Sample |
||  |  |
| --- | --- |
| Well 1 | DNA ladder |
| Well 2 | Colony 1 (Original Primers, Tm = 62°C ) |
| Well 3 | Colony 1 (Control Primers, Tm = 55°C ) |
| Well 4 | Colony 2 (Original Primers, Tm = 65°C ) |
| Well 5 | Colony 2 (Control Primers, Tm = 55°C ) |
| Well 6 | Control Plasmid |

***Table 4.** Colony PCR gel well setup.*

According to Figure 22, Colony 1 seemed to have our recombinant vector, as we could see bands of approximately 2 kB size, which matched with the weight of our insert ~ 2kB. However, we were unable to grow colonies from the successful DH5alpha transformation plate. We decided to discontinue our lab efforts on the SF9 expression aspect of our project because of time constraints and diverted our entire focus and resources on the *E. coli* expression aspect of our project. Results from the colony PCR indicated presence of the recombinant vector in Colony 1, however, we decided that this alone was not a definitive proof of successful cloning, as we had to discontinue our lab experiments for SF9. We were successful in optimizing the PCR conditions for our GC rich insert and plasmid sequences, and possibly also optimized conditions for the Ligation Independent Cloning.

---

### Conclusion

Overall, our goal was to design two expression systems in SF9 and Escherichia coli that would prove effective in allowing us to produce our protein of interest, protein S. After hours of foundational work, the designs detailed on our [design](https://2022.igem.wiki/stony-brook/design) page allowed us to accomplish this. Our plasmids and the genetic circuits were chosen and engineered to be best-suited for our experiments. Moreover, we assessed the efficiency of our gene circuits by modeling them in MATLAB and used the analytical data to determine our wet lab experiments. We confirmed successful insertion of the PROS1 gene into the *E. coli* expression vector 2Bc-T by amplifying the insert site using PCR and verifying its correct size using gel electrophoresis. BL21 and origami cell transformants were used for expression tests and the results confirm protein S expression from origami B cells.

We were unable to carry on our wet lab experiments for SF9 expression systems because of the time constraint imposed due to the complexity of the baculovirus expression system. However, we believe that our protocols, experimental data, parts collection and mathematical modeling data for SF9 expression systems can be used and improved upon by future iGEM teams to achieve successful expression of recombinant proteins from the baculovirus system. We hope that all the data and documentation of our experimental design on this page, and on our parts page, will be extremely useful for future research and scaling up protein S production as a therapeutic approach. We hope future iGEM teams and researchers looking into this and similar issues, will be able to use our designs as a foundation for their work.

![](https://static.igem.wiki/teams/4235/wiki/sidebarlogo.png)
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### Discover

[![](https://static.igem.wiki/teams/4235/wiki/footers/footer-1.png)](https://2022.igem.wiki/stony-brook/team)

## Team

The members of the 2022 Stony Brook University iGEM Team

[Learn More](https://2022.igem.wiki/stony-brook/team)

[![](https://static.igem.wiki/teams/4235/wiki/footers/footer-3.png)](https://2022.igem.wiki/stony-brook/parts)

## Description

Introduction to our project.

[Learn More](https://2022.igem.wiki/stony-brook/parts)

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