iGEM 2019 — Team UChicago

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UChicago/Notebook

[Home](https://2019.igem.org/Team%3AUChicago)

Team

[Team Members](https://2019.igem.org/Team%3AUChicago/Team)
[Collaborations](https://2019.igem.org/Team%3AUChicago/Collaborations)
[Public Engagement](https://2019.igem.org/Team%3AUChicago/Public_Engagement)

Project

[Description](https://2019.igem.org/Team%3AUChicago/Description)
 [Design](https://2019.igem.org/Team%3AUChicago/Design)
[Modeling](https://2019.igem.org/Team%3AUChicago/Model)
[Experiments](https://2019.igem.org/Team%3AUChicago/Experiments)
[Results](https://2019.igem.org/Team%3AUChicago/Results)
 [Future Direction](https://2019.igem.org/Team%3AUChicago/Improve)
 [Notebook](https://2019.igem.org/Team%3AUChicago/Notebook)
[References](https://2019.igem.org/Team%3AUChicago/References)
[Attributions](https://2019.igem.org/Team%3AUChicago/Attributions)

[Human Practices](https://2019.igem.org/Team%3AUChicago/Human_Practices)

Parts

[Design Goals](https://2019.igem.org/Team%3AUChicago/Parts)
[Basic Parts](https://2019.igem.org/Team%3AUChicago/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AUChicago/Composite_Part)
[Parts Collection](https://2019.igem.org/Team%3AUChicago/Part_Collection)

[Safety](https://2019.igem.org/Team%3AUChicago/Safety)

## Notebook


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UChicago/Experiments

[Home](https://2019.igem.org/Team%3AUChicago)

Team

[Team Members](https://2019.igem.org/Team%3AUChicago/Team)
[Collaborations](https://2019.igem.org/Team%3AUChicago/Collaborations)
[Public Engagement](https://2019.igem.org/Team%3AUChicago/Public_Engagement)

Project

[Description](https://2019.igem.org/Team%3AUChicago/Description)
 [Design](https://2019.igem.org/Team%3AUChicago/Design)
[Modeling](https://2019.igem.org/Team%3AUChicago/Model)
[Experiments](https://2019.igem.org/Team%3AUChicago/Experiments)
[Results](https://2019.igem.org/Team%3AUChicago/Results)
 [Future Direction](https://2019.igem.org/Team%3AUChicago/Improve)
 [Notebook](https://2019.igem.org/Team%3AUChicago/Notebook)
[References](https://2019.igem.org/Team%3AUChicago/References)
[Attributions](https://2019.igem.org/Team%3AUChicago/Attributions)

[Human Practices](https://2019.igem.org/Team%3AUChicago/Human_Practices)

Parts

[Design Goals](https://2019.igem.org/Team%3AUChicago/Parts)
[Basic Parts](https://2019.igem.org/Team%3AUChicago/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AUChicago/Composite_Part)
[Parts Collection](https://2019.igem.org/Team%3AUChicago/Part_Collection)

[Safety](https://2019.igem.org/Team%3AUChicago/Safety)

## Experiments

#### Cloning

**Goals:** To successfully produce the plasmids outlined in our design goals.
**Workflow:** Quick-change PCR was used to repair the Fatty acid photodecarboxylase gene which had a single base pair deletion when the gene was received from IDT. This gene was then inserted into a Psb1C3 backbone using gibson assembly.

PCR was used to amplify the presence of individual enzyme genes for FabF, FabD, FabZ and FabG from the FASyn Operon which were each individually inserted into pSB1C3 backbones using gibson assembly.
Quick-Change PCR was used to edit the SigA promoter to be active in E. Coli. PCR was used to add FLAG tags to the AlkL gene and SigA promoter, then the parts were reassembled into pSB1C3 using Gibson Assembly.
Many difficulties were encountered during our attempts at gibson assembly due to homology between the biobrick prefix and suffix, so a new set of primers that could replace the old set of standardized primers (VR,VF2, suffix adaptor and prefix adaptor) was created. See our [results page](https://2019.igem.org/Team%3AUChicago/Results) to learn more.

#### Growth Curves

**Goals:** The growth curve data was taken to empirically measure the growth rates of Synechococcus in several conditions and implicitly determine some of the effects of the transformation of our plasmids. We took data on a control sample, a sample with the fatty acid photodecarboxylase gene inserted into a backbone with chloramphenicol resistance (Alk), a sample with the AlkL channel protein gene inserted into a backbone with ampicillin resistance (AlkL), and a sample with both plasmids transformed and selected for by both antibiotics.
**Protocol:**
**1.** Transform desired plasmids into E. coli and plate them.
**2.** Make overnight cultures, with 3 or 4 replicates for each plasmid.
**3.** The following day, take the OD600 of each culture. Make an approximately 10X dilution to get all of the cultures down to a similar OD.
**4.** Take a starting OD.
**5.** Incubate the diluted cultures in a shaker at 37°C under blue light for 2 hours.
**6.** After 2 hours, take the OD again.
**7.** Continue to take the OD every 2 hours.

#### Gas Chromatography Mass Spectrometry

**Goals:** The goal for this procedure was to assess the production of alkanes in E. Coli containing the Alk Operon.
**Protocol:** The Alk-operon was transformed into E.coli and those E.coli were grown in an LB broth overnight in a photosynthetic incubator where they were kept overnight at 37C and exposed to a white light source for the duration of the night. The overnight cultures were set for 5 days and stored at room temperature. Next the overnight cultures were centrifuged down and the pellets were resuspended in in 350 ml of STET buffer (10 mM Tris-HCl, pH 8.0, with 0.1 M NaCl, 1 mM EDTA, and 5% [w/v] TRITON“ X-100). Next, 25 ml of a freshly prepared lysozyme solution (10 mg/ml in 10 mM Tris-HCl, pH 8.0) was added to the samples. After a 30 minute incubation at 37C, the lysis mixture was centrifuged at 14,000 Xg for 10 minutes.
The Gas Chromatography Mass Spectroscopy (GCMS) was performed with the solvent Hexane. 200ul of Hexane was added to lysis mixture and then 100ul of that new mixture was pipetted into a vial containing another 900ul of Hexane. This sample was compared to a negative control in which the identical procedure was carried out on transformed E. coli with a PSB1C3 empty vector. The sample was also compared to a positive control that contained 80ul of an Alkane standard ladder dissolved in 400uL. All three samples were run through Agilent 7200B Q-Tof GCMS and the results were analyzed to assess the production of alkanes in the E. coli containing the Alk Operon.

#### Fluorescence Assay

**Goals:** The goal of our fluorescence assays was to characterize the ribosome binding site with part number BBa\_J61100. The specific goal was to characterize green fluorescent protein expression in *E. Coli*
**Protocol:** The protocol followed was developed in iGEM's 2018 interlab study, and can be found [here.](https://2019.igem.org/Measurement/Protocols)

#### Western Blot

**Goals:** Western Blots were performed to ensure proper expression of the AlkL protein. AlkL was the only FLAG-tagged protein in this experiment.
**Protocol:**

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UChicago/Results

[Home](https://2019.igem.org/Team%3AUChicago)

Team

[Team Members](https://2019.igem.org/Team%3AUChicago/Team)
[Collaborations](https://2019.igem.org/Team%3AUChicago/Collaborations)
[Public Engagement](https://2019.igem.org/Team%3AUChicago/Public_Engagement)

Project

[Description](https://2019.igem.org/Team%3AUChicago/Description)
 [Design](https://2019.igem.org/Team%3AUChicago/Design)
[Modeling](https://2019.igem.org/Team%3AUChicago/Model)
[Experiments](https://2019.igem.org/Team%3AUChicago/Experiments)
[Results](https://2019.igem.org/Team%3AUChicago/Results)
 [Future Direction](https://2019.igem.org/Team%3AUChicago/Improve)
 [Notebook](https://2019.igem.org/Team%3AUChicago/Notebook)
[References](https://2019.igem.org/Team%3AUChicago/References)
[Attributions](https://2019.igem.org/Team%3AUChicago/Attributions)

[Human Practices](https://2019.igem.org/Team%3AUChicago/Human_Practices)

Parts

[Design Goals](https://2019.igem.org/Team%3AUChicago/Parts)
[Basic Parts](https://2019.igem.org/Team%3AUChicago/Basic_Part)
[Composite Parts](https://2019.igem.org/Team%3AUChicago/Composite_Part)
[Parts Collection](https://2019.igem.org/Team%3AUChicago/Part_Collection)

[Safety](https://2019.igem.org/Team%3AUChicago/Safety)

## Results

**Characterize a Part [BBa\_J61100](https://http://parts.igem.org/Part%3ABBa_J61100)**:
The Strength of this ribosome binding site has previously been shown to be extremely week, but up to this point, it has not been evaluated using the protocol that was designed as a result of the 2018 Interlab Study. UChicago set out to characterize this RBS using the Interlab Protocol. To do this, we attached this promoter RBS construct to [BBa\_I746916](https://http://parts.igem.org/Part%3ABBa_I746916) which is a super folder GFP. We then followed the Protocol for a plate reader experiment using this construct, a blank pSB1C3 vector, and a blank non-iGEM vector.

From the plot below, we can definitely see that there are small amounts of green fluorescent protein (gfp) present in our ligation sample. We have chosen to use fluorescent measurement as a means to measure our ribosome binding site efficiency because the ribosome binding sites connect the promoter with an iGEM gfp. Since there is gfp present, this means that our characterization was successful. Our empty vector control shows small amounts of gfp due to the nature of our promoter BBa\_J23119. Our negative control shows similar readings as the background readings on our plate reader, which means that our negative control is successful because there are no gfp present. This serves as a good reference for our sample comparison. The finding that there is no significant difference between our negative control and the gfp part in fluorescence confirms [team Delft's measurements that this ribosome binding site is weak(found here).](http://parts.igem.org/Part%3ABBa_J61100) Furthermore, a non-iGEM vector [(adapted from Rauch et al, 2019)](https://www.sciencedirect.com/science/article/pii/S0092867419306208) showed lower fluorescence than did the iGEM vector with and without the gfp gene inserted. This indicates some auto-fluorescence of the iGEM vector which limits the feasibility of the [2018 interlab protocol](https://2019.igem.org/Measurement/Protocols) when characterizing the fluorescence of low efficiency parts.

![](https://static.igem.org/mediawiki/2019/3/3d/T--UChicago--gfpplot.png)

**FASyn Operon:** For the FASyn operon, we encountered many errors with gibson assembly and ultimately decided to create individual plasmid constructs for many of the relevant genes in the Fatty acid synthesis pathway. This would enable us to individually troubleshoot any issues with each individual gene and also provided the necessary groundwork for our future directions with this project. Below are the chromatograms showing DNA sequencing results which indicate successful creation of FabD, FabG, and FabF. Note that FabG was not assembled into a plasmid and thereby is only a gene fragment whereas FabD and FabF were assembled into plasmids with promoters and ribosome binding sites.

**Fab D Chromatogram**

![](https://static.igem.org/mediawiki/2019/6/66/T--UChicago--FabDChromatogram.jpeg)

**Fab F Chromatogram**

![](https://static.igem.org/mediawiki/2019/7/78/T--UChicago--fabFchromatogram.jpeg)

**Fab G Chromatogram**

![](https://static.igem.org/mediawiki/2019/3/38/T--UChicago--fabgchromatogram.png)

**Alk Operon:** To validate the functionality of the alkane part, we tested our part in E coli rather than Synechococus due to the growth time difference. Growth curve experiments demonstrated that cells with the Alk operon transformed grew at a significantly slower rate compared to their control (see exporter operon results below). This suggests that the Alk part is mildly toxic to the cells which corresponds to its predicted effect of producing alkanes. To further confirm this was due to a functionality of the Alk part rather than the protein itself, we performed Gas Chromatography Mass spectrometry to confirm the existence of alkanes. It was clear that cells that were allowed to grow overnight in the presence of white light to provide the blue light needed to activate the enzyme. The gas chromatogram, when compared to control, clearly indicated a difference in composition (Figure 2a), but a search of the NIST Mass Spec Library suggested that this was Triton-X, not the alkanes we originally thought (Figure 2b).

![](https://static.igem.org/mediawiki/2019/6/6e/T--UChicago--fig2a1.png)![](https://static.igem.org/mediawiki/2019/f/f7/T--UChicago--fig2a2.png)

**Figure 2a**: Comparison of Gas Chromatograms of hexane cell extract from FAP cells (bottom) and negative control (top)

![](https://static.igem.org/mediawiki/2019/7/7c/T--UChicago--fig2b1.png)![](https://static.igem.org/mediawiki/2019/a/a4/T--UChicago--fig2b2.png)

**Figure 2b**: Mass Spectrum of retention time 6-6.2 min of FAP cells (bottom). Spectrum of Negative Control (top)

It wasn’t until one lab member forgot to clean up samples and left them out for 5 days that different results began to emerge. After 5 days at RT, all of the negative control solutions had turned pink while the supernatants transformed with the Alk Operon remained white as during the initial preps (Figure 3). Once Again, GCMS analysis was run on both samples after hexane extraction. The Chromatograms do not indicate a distinct difference (Figure 4a). However, when Mass Spec plots were generated at the 6.1-6.2 retention time, the composition of those plots was quite different. The sample chromatogram showed characteristic peaks produced by long chain alkanes while the control peaks were more characteristic of the previously observed Triton-X (Figure 4b). Search of the NIST Mass Spec Library top 9 hits further supported these results ([Report 1](https://static.igem.org/mediawiki/2019/7/75/T--UChicago--Report1.pdf)). This demonstrates that our Fatty Acid Photocarboxylase enzyme works in converting fatty acids in the cells to alkanes, thereby allowing for efficient biofuels production.

![](https://static.igem.org/mediawiki/2019/0/0a/T--UChicago--fig3.jpeg)

**Figure 3**: Photographs of samples that were left out for 5 days at RT. FAP sample (Left) and Negative control (Right)

![](https://static.igem.org/mediawiki/2019/e/e7/T--UChicago--fig4a1.png)![](https://static.igem.org/mediawiki/2019/f/f8/T--UChicago--fig4a2.png)![](https://static.igem.org/mediawiki/2019/9/9b/T--UChicago--fig4a3.png)![](https://static.igem.org/mediawiki/2019/4/4f/T--UChicago--fig4a4.png)

**Figure 4a**: Gas Chromatograms of samples after 5 days. FAP (Top two) and negative control Bottom (Bottom two)

![](https://static.igem.org/mediawiki/2019/8/82/T--UChicago--fig4b1.png)![](https://static.igem.org/mediawiki/2019/5/5b/T--UChicago--fig4b2.png)

**Figure 4b**: Mass Spectrum of time point 6.1-6.2 of FAP (left) and negative control (Right) critical

![](https://static.igem.org/mediawiki/2019/a/ab/T--UChicago--fig4c.png)

**Figure 4c**: Mass Spectrum of time point 6.0 of Alkane Standard (Sigma Aldrich)

**Exporter Operon:** The exporter operon was the only plasmid that was properly synthesized and FLAG-tagged for Western Blot. A sequencing chromatogram can be seen below.

![](https://static.igem.org/mediawiki/2019/4/41/T--UChicago--AlkLchromatogram.png)

We constructed growth curves to test the exporter operon's impact on growth rate both with and without the presence of the Alk Operon. As is shown below, growth with the Exporter (AlkL) operon seems to follow that of the control quite closely. The Alk Operon, however, ends at a lower plateau, which is likely due to alkane toxicity. Growth with both operons plateaus with an even lower optical density. A possible reason for this lower plateau is the presence of two antibiotics (ampicillin and chloramphenicol).

![](https://static.igem.org/mediawiki/2019/0/0d/T--UChicago--GrowthCurve1.png)
![](https://static.igem.org/mediawiki/2019/5/5f/T--UChicago--growthbarsalkL.png)

To test the effect of two antibiotics, a secondary growth curve was constructed. This secondary growth curve test compares cells with both Alk and AlkL operons to control cells with empty vectors with both antibiotic resistances. The cells with both operons reach a lower plateau than that of the control cells. This suggests that despite being an importer, AlkL's passivity does allow for alkanes to exit the cells when the intracellular concentration reaches a high enough point. For the Alk+AlkL cells though, this point is reached when the alkane levels within the cell are already too toxic, and growth has slowed.

![](https://static.igem.org/mediawiki/2019/a/a7/T--UChicago--GrowthCurve2.jpeg)

Furthermore, we performed a Western Blot to validate the expression of our channel protein in E. Coli. As is shown in the image below, we were able to detect a faint band of the proper size - 26 kDa.

![](https://static.igem.org/mediawiki/parts/a/a6/T--UChicago--WesternBlotAlkL.jpeg)