iGEM 2017 — Team ColumbiaNYC

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# Team:ColumbiaNYC/Notebook

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* [![](https://static.igem.org/mediawiki/2017/f/f3/T--ColumbiaNYC--columbiauniversity-white-crown.jpg)
  COLUMBIA UNIVERSITY](https://2017.igem.org/Team%3AColumbiaNYC)
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  + [DESCRIPTION](https://2017.igem.org/Team%3AColumbiaNYC/Description)
  + [PROOF OF CONCEPT](https://2017.igem.org/Team%3AColumbiaNYC/Design)
  + [RESULTS](https://2017.igem.org/Team%3AColumbiaNYC/Results)
  + [SAFETY](https://2017.igem.org/Team%3AColumbiaNYC/HP/Silver)
  + [INTERLAB](https://2017.igem.org/Team%3AColumbiaNYC/InterLab)
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  + [FUTURE WORK](https://2017.igem.org/Team%3AColumbiaNYC/Improve)
* [PARTS](https://2017.igem.org/Team%3AColumbiaNYC/Part_Collection)
  + [BASIC PARTS](https://2017.igem.org/Team%3AColumbiaNYC/Basic_Part)
  + [COMPOSITE PARTS](https://2017.igem.org/Team%3AColumbiaNYC/Composite_Part)
  + [PARTS COLLECTION](https://2017.igem.org/Team%3AColumbiaNYC/Part_Collection)
* [MODELLING](https://2017.igem.org/Team%3AColumbiaNYC/Model)
* [NOTEBOOK](https://2017.igem.org/Team%3AColumbiaNYC/Notebook)
  + [BRAINSTORM](https://2017.igem.org/Team%3AColumbiaNYC/Brainstorm)
  + [LAB NOTEBOOK](https://2017.igem.org/Team%3AColumbiaNYC/Notebook)
* [HUMAN PRACTICES](https://2017.igem.org/Team%3AColumbiaNYC/Engagement)
  + [HUMAN PRACTICES](https://2017.igem.org/Team%3AColumbiaNYC/HP/Gold_Integrated)
  + [ENGAGEMENT](https://2017.igem.org/Team%3AColumbiaNYC/Engagement)
  + [ENTREPRENEURSHIP](https://2017.igem.org/Team%3AColumbiaNYC/Entrepreneurship)
  + [COLLABORATIONS](https://2017.igem.org/Team%3AColumbiaNYC/Collaborations)
  + [MEDAL CRITERION](https://2017.igem.org/Team%3AColumbiaNYC/Medals)

# Lab Notebook

Email: cuigem2017team@googlegroups.com

[![](https://static.igem.org/mediawiki/2017/e/e4/T--ColumbiaNYC--columbiauniversity.jpg)](http://www.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/e/ee/T--ColumbiaNYC--cusystemsbiology.jpg)](https://systemsbiology.columbia.edu/)
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# Team:ColumbiaNYC/Experiments

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* [![](https://static.igem.org/mediawiki/2017/f/f3/T--ColumbiaNYC--columbiauniversity-white-crown.jpg)
  COLUMBIA UNIVERSITY](https://2017.igem.org/Team%3AColumbiaNYC)
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  + [INTERLAB](https://2017.igem.org/Team%3AColumbiaNYC/InterLab)
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* [HUMAN PRACTICES](https://2017.igem.org/Team%3AColumbiaNYC/Engagement)
  + [HUMAN PRACTICES](https://2017.igem.org/Team%3AColumbiaNYC/HP/Gold_Integrated)
  + [ENGAGEMENT](https://2017.igem.org/Team%3AColumbiaNYC/Engagement)
  + [ENTREPRENEURSHIP](https://2017.igem.org/Team%3AColumbiaNYC/Entrepreneurship)
  + [COLLABORATIONS](https://2017.igem.org/Team%3AColumbiaNYC/Collaborations)
  + [MEDAL CRITERION](https://2017.igem.org/Team%3AColumbiaNYC/Medals)

# Protocols

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### [Bacteria Chemical Transformation](#collapseOne)

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2. Remove agar plates(containing chloramphenicol) from storage at 4°C and let warm up to
   room temperature and then (optional) incubate in 37°C incubator. Alternatively these can
   be made, by adding roughly 15 mL of LB per plate with about 1 microliter per mL of 25
   microgram/mL working stock of chloramphenicol.
3. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 50 μL of competent cells in a
   microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your
   finger a few times.

**Note:**​​ Transformation efficiencies will be approximately 10-fold lower for ligation of
inserts to vectors than for an intact control plasmid.

4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a
   42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on
   the competent cells you are using). In our case, we used
   a dry bath for 45 seconds which proved to be just as
   effective.
6. Put the tubes back on ice for 2 min.
7. Add 500 μl LB or SOC media (without antibiotic) to the
   bacteria and grow in 37°C shaking incubator for 45 min.

**Note:**​ This outgrowth step allows the bacteria time to
generate the antibiotic resistance proteins encoded
on the plasmid backbone so that they will be able to
grow once plated on the antibiotic containing agar
plate. This step is not critical for Ampicillin resistance
but is much more important for other antibiotic
resistances.

8. Plate 100 microliters of the transformation onto a 10 cm
   LB agar platecontaining the chloramphenicol. The
   bacteria can be spread via beads, quad streaking, or a spreader. Any method works.

**Note:**​ We recommend that you plate 50 μL on one plate and the rest on a second
plate. This gives the best chance of getting single colonies, while allowing you to
recover all transformants.

**Note:**​ If the culture volume is too big, gently collect the cells by centrifugation and
resuspend in a smaller volume of LB so that there isn't too much liquid media on the
agar plates. If the agar plate doesn't dry adequately before the cells begin dividing,
the bacteria diffuse through the liquid and won't grow in colonies.

9. Incubate plates at 37°C overnight.

### [Attribution #2](#collapseTwo)

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

### [Attribution #3](#collapseThree)

Anim pariatur cliche reprehenderit, enim eiusmod high life accusamus terry richardson ad squid. 3 wolf moon officia aute, non cupidatat skateboard dolor brunch. Food truck quinoa nesciunt laborum eiusmod. Brunch 3 wolf moon tempor, sunt aliqua put a bird on it squid single-origin coffee nulla assumenda shoreditch et. Nihil anim keffiyeh helvetica, craft beer labore wes anderson cred nesciunt sapiente ea proident. Ad vegan excepteur butcher vice lomo. Leggings occaecat craft beer farm-to-table, raw denim aesthetic synth nesciunt you probably haven't heard of them accusamus labore sustainable VHS.

Email: cuigem2017team@googlegroups.com

[![](https://static.igem.org/mediawiki/2017/e/e4/T--ColumbiaNYC--columbiauniversity.jpg)](http://www.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/e/ee/T--ColumbiaNYC--cusystemsbiology.jpg)](https://systemsbiology.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/8/84/T--ColumbiaNYC--columbiauniversity-seas.jpg)](https://engineering.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/c/cd/T--ColumbiaNYC--columbiauniversity-cumc.jpg)](http://www.cumc.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/8/85/T--ColumbiaNYC--columbiauniversity-path.jpg)](http://pathology.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/a/aa/T--ColumbiaNYC--columbiauniversity-cs.jpg)](https://www.cs.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/9/99/T--ColumbiaNYC--columbiauniversity-biological-sciences.jpg)](http://biology.columbia.edu/)
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# Team:ColumbiaNYC/Results

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  COLUMBIA UNIVERSITY](https://2017.igem.org/Team%3AColumbiaNYC)
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  + [PROOF OF CONCEPT](https://2017.igem.org/Team%3AColumbiaNYC/Design)
  + [RESULTS](https://2017.igem.org/Team%3AColumbiaNYC/Results)
  + [SAFETY](https://2017.igem.org/Team%3AColumbiaNYC/HP/Silver)
  + [INTERLAB](https://2017.igem.org/Team%3AColumbiaNYC/InterLab)
  + [APPLIED DESIGN](https://2017.igem.org/Team%3AColumbiaNYC/Applied_Design)
  + [FUTURE WORK](https://2017.igem.org/Team%3AColumbiaNYC/Improve)
* [PARTS](https://2017.igem.org/Team%3AColumbiaNYC/Part_Collection)
  + [BASIC PARTS](https://2017.igem.org/Team%3AColumbiaNYC/Basic_Part)
  + [COMPOSITE PARTS](https://2017.igem.org/Team%3AColumbiaNYC/Composite_Part)
  + [PARTS COLLECTION](https://2017.igem.org/Team%3AColumbiaNYC/Part_Collection)
* [MODELLING](https://2017.igem.org/Team%3AColumbiaNYC/Model)
* [NOTEBOOK](https://2017.igem.org/Team%3AColumbiaNYC/Notebook)
  + [BRAINSTORM](https://2017.igem.org/Team%3AColumbiaNYC/Brainstorm)
  + [LAB NOTEBOOK](https://2017.igem.org/Team%3AColumbiaNYC/Notebook)
* [HUMAN PRACTICES](https://2017.igem.org/Team%3AColumbiaNYC/Engagement)
  + [HUMAN PRACTICES](https://2017.igem.org/Team%3AColumbiaNYC/HP/Gold_Integrated)
  + [ENGAGEMENT](https://2017.igem.org/Team%3AColumbiaNYC/Engagement)
  + [ENTREPRENEURSHIP](https://2017.igem.org/Team%3AColumbiaNYC/Entrepreneurship)
  + [COLLABORATIONS](https://2017.igem.org/Team%3AColumbiaNYC/Collaborations)
  + [MEDAL CRITERION](https://2017.igem.org/Team%3AColumbiaNYC/Medals)

# Results

Assessment of shRNA knockdown

### **shRNA Design**

We chose an shRNA sequenced published on Addgene to knockdown our eGFP, and we compared the shRNA sequence to the eGFP
DNA sequence to make sure the knockdown will theoretically occur. To construct the plasmid, we assembled the shRNA-encoding
DNA from two segments that made up the sequence because the natural hairpin structure in the shRNA made the full sequence
difficult to synthesize in one piece. Because our project design relies on the shRNA to inhibit gene expression (ultimately
oncogene expression) and shRNA is not extremely stable (but not unstable), the bacteria must produce large quantities
of shRNA for this gene therapy approach to be effective. Thus, we chose to put the gene in a pUC plasmid, which is
a very high-copy plasmid (500-600 copies per cell). To increase the production of shRNA further, we placed the shRNA
production under the control of a T7 promoter, which causes very strong gene expression compared to other promoters.
Our final shRNA plasmid design is the following:

![](https://static.igem.org/mediawiki/2017/4/4d/T--ColumbiaNYC--silenshr.png)

###### **Figure 1:** The SilenshR plasmid. This is an IPTG-inducible circuit with a T7 promoter that produces an shRNA that knocks down commercialized eGFP.

### Transformation of Plasmid into BL21 (DE3) *E.coli*

We transformed this plasmid into BL21 (DE3) E. coli cells and got three colonies. To confirm that cloning was successful,
we first performed colony PCR to determine if the insert was successfully cloned into the backbone. As can be seen,
the colony PCR results show that the insert was successfully cloned in each of the three colonies:

![](https://static.igem.org/mediawiki/2017/b/bd/Columbia_university_gel.jpg)

###### **Figure 2:** Lane 1 is the Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb) from NEB, lane 2 is the negative control, and lane 3 through 5 are from the three transformed colonies. The bands in lanes 3 through 5 are about 500bp, which is what we expect, and they are different in size from the negative control. Thus, this shows that cloning was successful.

### Sequencing our Results

We then performed Sanger sequencing on each colony to ensure that the shRNA sequence in the plasmid is correct, and analysis
of the results showed that it is correct.

![](https://static.igem.org/mediawiki/2017/8/8d/Columbia_university_seqdata2.jpg)

###### **Figure 3:** Sanger sequencing results for the insert for the 2 colonies

### GFP Expression Results

We then produced the shRNA in vitro using the New England Biolabs T7 expression kit to use in the lipofectamine assay.
After the in vitro shRNA is successful, we reran the lipofectamine assay with shRNA that was produced in vivo using
IPTG induction. The shRNA produced in vivo was isolated using the miRNeasy kit. We extracted a significant quantity
of shRNA using both in vitro expression and in vivo expression. In in vitro expression, the concentration of shRNA
was about 2230 ng/uL. In in vivo expression, concentrations ranged from 850 ng/uL to 1070 ng/uL. The lipofectamine
assay would test whether the shRNA successfully knocks down the eGFP in mammalian cells. In this assay, the shRNA would
be introduced into liposomes, and then the liposomes would be introduced into the mammalian cell line. The liposomes
would fuse with the cell membrane of the mammalian cells and then the shRNA would enter the cells. Upon entry, the
shRNA would knock down the expression of eGFP. The effectiveness of the knockdown of eGFP from the shRNA is measured
using a flow cytometer. The flow cytometer measures the GFP florescence in every cell and gives a statistical distribution
of the florescence. The results from the synthesized shRNA is compared to a negative control, where none of the shRNA
is introduced to the mammalian cells during the lipofectamine assay. The results will also be compared to a positive
control, where we used siRNA designed and proven to knock down eGFP from Thermo Fisher. We analyzed the decrease in
florescence to determine the effectiveness of the shRNA-mediated knockdown of GFP. (For shRNA synthesized to knockdown
oncogenes, we plan to use RT-qPCR to detect knockdown of the target gene. For the RT-qPCR, we will use the reverse
transcriptase to transcribe the mRNA produced by the bacteria into cDNA. Then in qPCR, we can determine the amount
of mRNA remaining in the mammalian cells from the rate at which qPCR proceeds.)

![](https://static.igem.org/mediawiki/2017/8/8b/Columbia_university_biobrickgraph.jpg)

###### **Figure 4:** The x-axis shows the negative control (PBS), the positive controls (siRNA), and the experimental shRNA designed to inhibit eGFP (shRNA). The y-axis shows the percentage of mammalian cells that were fluorescing eGFP (defined as eGFP positive). The error bars are 2 standard error, or a 95% confidence interval. As can be seen, the confidence intervals for the negative control and the experimental shRNA do not overlap, which shows that this difference is statistically significant. This indicates that our shRNA successfully knocks down eGFP expression in HeLa cells. Hard data used to generate this graph can be found [here](https://docs.google.com/a/columbia.edu/spreadsheets/d/18TKvTeAUxyy6XsE-lLb64fjqCos5Lhl2_f7p8wQLM4I/edit?usp=sharing).

These results show that our proof-of-concept works successfully, where shRNA successfully knocks down expression of the
targeted gene.

Email: cuigem2017team@googlegroups.com

[![](https://static.igem.org/mediawiki/2017/e/e4/T--ColumbiaNYC--columbiauniversity.jpg)](http://www.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/e/ee/T--ColumbiaNYC--cusystemsbiology.jpg)](https://systemsbiology.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/8/84/T--ColumbiaNYC--columbiauniversity-seas.jpg)](https://engineering.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/c/cd/T--ColumbiaNYC--columbiauniversity-cumc.jpg)](http://www.cumc.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/8/85/T--ColumbiaNYC--columbiauniversity-path.jpg)](http://pathology.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/a/aa/T--ColumbiaNYC--columbiauniversity-cs.jpg)](https://www.cs.columbia.edu/)
[![](https://static.igem.org/mediawiki/2017/9/99/T--ColumbiaNYC--columbiauniversity-biological-sciences.jpg)](http://biology.columbia.edu/)
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