iGEM 2016 — Team ColumbiaU_NYC

[![](https://static.igem.org/mediawiki/2016/8/8b/HQ_page_logo.jpg)](https://2016.igem.org)

# Team:ColumbiaU NYC/Notebook

<!DOCTYPE html>
Columbia University iGEM

![Columbia Logo](https://static.igem.org/mediawiki/2016/3/32/Igem-logo.png)

* [Home](https://2016.igem.org/Team%3AColumbiaU_NYC)
* [About](https://2016.igem.org/Team%3AColumbiaU_NYC/About)
* [People](https://2016.igem.org/Team%3AColumbiaU_NYC/Team)
* [Parts](https://2016.igem.org/Team%3AColumbiaU_NYC/Parts)
* Documentation
  + [Project](https://2016.igem.org/Team%3AColumbiaU_NYC/Project)
  + [Notebook](https://2016.igem.org/Team%3AColumbiaU_NYC/Notebook)
  + [Design](https://2016.igem.org/Team%3AColumbiaU_NYC/Design)
  + [Proof of Concept](https://2016.igem.org/Team%3AColumbiaU_NYC/Proof)
  + [Demonstrate](https://2016.igem.org/Team%3AColumbiaU_NYC/Demonstrate)
  + [Description](https://2016.igem.org/Team%3AColumbiaU_NYC/Description)
* [Acknowledgements](https://2016.igem.org/Team%3AColumbiaU_NYC/Attributions)
* [Sponsors](https://2016.igem.org/Team%3AColumbiaU_NYC/Sponsors)
* [Outreach](https://2016.igem.org/Team%3AColumbiaU_NYC/Engagement)

* [May](#may)
  + [5/22-5/28](#5-22)
  + [5/29-6/4](#5-29)
* [June](#june)
  + [6/5-6/11](#6-5)
  + [6/12-6/18](#6-12)
  + [6/19-6/25](#6-19)
  + [6/26-7/2](#6-26)
* [July](#july)
  + [7/3-7/9](#7-3)
  + [7/10-7-16](#7-10)
  + [7/17-7/23](#7-17)
  + [7/24-7/30](#7-24)
  + [7/24-7/30](#7-24)
  + [7/31-8/6](#7-31)
* [August](#August)
  + [8/7-8/13](#8-7)
  + [8/14-8/20](#8-14)
  + [8/21-8/27](#8-21)
  + [8/28-9/3](#8-28)
* [September](#September)
  + [9/4-9/10](#9-4)
  + [9/11-9/17](#9-11)
  + [9/18-9/24](#9-18)
  + [9/25-10/1](#9-25)

# May

---

## 5/22-5/28

---

We solidified the synthetic mosquito repellent
idea. We spent the week conducting a literature
review to determine what research has been
conducted on the rhamnolipids and bacteria
strains. We pinpointed a few subtopics that we
could pursue such as freeze drying and skin
grafting. We divided the research among ourselves
and put together a powerpoint containing a summary
of our ideas to present to our faculty
advisors. After the meeting, we made a
comprehensive project timeline schedule and made
experimental plans for the following week.

## 5/29-6/4

---

#### Molecular Cloning

We started molecular cloning with a PCR amplification of
the RhlAB operon from the P. aeruginosa P14 genome
extract. For the screening assay, we used a superfolded
GFP. With our faculty members, we discussed whether we
should clone using EcoRI restriction enzyme cuts or Gibson
assembly. Initially, our PCR was unsuccessful because the
primers we designed had too few base pairs annealing to
the sequence. After ordering the correct primers, we used
Gibson assembly to insert the gene into the backbone,
which was successful. Additionally, we transformed
plasmids without the RhlAB insert into P. putida
KT2440. Then, we picked colonies with different GFP
intensities to obtain a library of different constitutive
promoters as well.

#### Qualitative Analysis of Rhamnolipids

Regarding the production of rhamnolipids, we researched a
few different tests before deciding to try the CTAB
qualitative test with pure rhamnolipids. Furthermore, we
decided to try freeze-dry *P. putida* to determine what
percent of the bacteria would be viable after extended
periods of time. We could use this knowledge to determine
shelf-life of the mosquito repellent for commercial
applications.

#### Mosquito and Mouse Experiments

We reached out to several researchers as well in
preparation for future experiments. We contacted
Dr. Voshall at Rockefeller University who does mosquito
testing. We want to determine what concentration of
mono-rhamnolipids and di-rhamnolipids actively repel
mosquitoes. We also reached out to Dr. Christiano who
works with mice to discuss whether we can use her
laboratory to conduct rhamnolipid experiments using human
skin grafted on mice. In preparation for the meeting, we
drafted a set of experimental protocols and created a
Powerpoint presentation.

#### Community Outreach

We planned a community outreach project in which we can
administer a survey to individuals to determine if they
are highly attracted or unattracted to mosquitoes and then
use next generation sequencing to sequence samples of the
skin biome to deduce if there are any commonalities.

# June

---

## 6/5-6/11

---

#### Freeze Dry

The initial freeze dry experiments were successful but we
realized that we need a more quantitative way to analyze
the success of the freeze dry. Perhaps, we need to
incorporate a survival rate or ratio calculation. We
decided that we need to also extend the timeline for the
freeze dry experiments to more closely mimic real world
situations.

#### Rhamnolipid Quantification

We conducted the CTAB experiments with the rhamnolipids
and blue halos were present. However, we realized CTAB
only works well for higher concentrations than what we
need to produce. Furthermore, we would need a more
quantitative metric than qualitative one. We brainstormed
and figured that we could use an image application to
measure the diameter of the halos and correlate that to
the concentration of rhamnolipids.

#### Community Outreach

We had several meetings this week one of which was with
Dr. Shaman, a skin swabbing expert, to discuss our idea
for community outreach. Dr. Shaman had collaborated with
the Museum of Natural History to swab skin biomes in the
skin biome exhibit. He suggested that we avoid
collaborating with the museum due to the extensive IRB
process and provisions of the board of trustees. He also
cautioned our timeline and monetary expenses.

#### Keratinocyte Experiments

We met with Dr. Prince who is a skin bacteria specialist
to discuss collaboration. We planned to conduct
experiments to determine how the bacteria strains and
rhamnolipids affect skin cell viability. Dr. Prince liked
our idea and put us in contact with a graduate student in
her laboratory to gather more information. She agreed to
provide her lab space, human skin cells (keratinocyte),
cell media, and other reagents to aid in our experiments.

## 6/12-6/18

---

#### Molecular Cloning

Our Gibson assembly worked for two different promoters; we
now have two constructs transformed in E. coli including a
strong promoter from Streptococcus pneumoniae and a weak
promoter from Propionibacterium acnes. We started to work
on the construction of BioBrick parts. We began to build
BioBricks for different promoters for the RhlAB operon. We
made plans to characterize the promoters in E. coli,
P. aeruginosa, and P. putida. This week, we also continued
experiments to transform recombinant plasmids into
P. putida using electroporation. We contacted Dr. Jiang
from the Marraffini Rockefeller University to obtain the
S. epidermidis strains, recommended plasmids, and shuttle
vector for transformation/conjugation. He also sent us
specific protocols to utilize with this strain.

#### Rhamnolipid Quantification

We conducted more CTAB experiments in which we made test
plates with different concentrations of rhamnolipids to
make a standard curve using the diameters of the halos for
our quantitative measure. Unfortunately, we determined
that the halo diameter varies with the thickness of the
CTAB on plates in that thicker plates give bigger halos
for the same concentration of rhamnolipids. This
characteristic created inconsistency across different CTAB
plates. There were no visible halos for low concentrations
of rhamnolipids indicating that we might need to calibrate
CTAB to detect lower concentrations of rhamnolipids. We
also conducted the CTAB test using live E. coli cells with
the RhlAB recombinant plasmid, but there were no visible
Halos possibly due to low Rhamnolipid concentrations. The
overall conclusion is that the CTAB test may be used as a
qualitative analysis, but is not reliable as a
quantitative method due to condition-dependent halo
diameters. We found a different assay called an orcinol
assay that may be used in place of the CTAB test.

#### Freeze Dry

We focused on counting the exact levels of survival using cell count methods.

#### Mosquito Experiments

Because the lab at Rockefeller University did not respond
to emails, we contacted the Fidock lab at Columbia
University. The Fidock lab has extensive experience with
Malaria experiments and has incubators that they would be
willing to allow us to use. We made a meeting for the
following week and began to draft experimental plans to
discuss with Professor Fidock in person.

## 6/19-6/25

---

#### Molecular Cloning

We received S. Epidermidis strain RP62A, shuttle strain
S. Aureus RN4220, a plasmid pC194 from S.A. RN4220, a
plasmid pC221 and pGO1 from S.A. OS2 from Dr. Jiang at
Rockefeller. We will start cloning next week once we
design the primers.

#### Freeze Dry

Experiments were conducted using M9 minimal media or LB as
the rehydration media. The lyoprotectants used was either
10% Sucrose or 10% Sucrose with LB. Based upon the
analysis conducted in the freeze dry research paper,
survival rate was calculated. The highest survival rate
calculated was 2.25% using LB as the rehydration and the
10% Sucrose lyoprotectants.

#### Rhamnolipid Quantification

As a way of detecting rhamnolipids, we decided to try
thin-layer chromatography (TLC). For different staining
methods, we tried orcinol with 50% sulfuric acid, orcinol
with 10% sulfuric acid, CAM, and KMnO4. We discovered that
orcinol with 10% sulfuric acid gives the best visibility,
and the limit of detection was determined to be 0.5
mg/mL. We were also able to distinguish between
mono-rhamnolipids and di-rhamnolipids through TLC
analysis.

#### Mosquito Experiments

We met with Professor Fidock who agreed to let us work in
his laboratory. He put us in contact with a mosquito
seller from NYU and a trained epidemiologist with whom we
can discuss the experimental setup. However, the
epidemiologist will not be available until August
1st. Thus, in the meantime, we will communicate via email
regarding the experimental protocol and setup.

## 6/26-7/2

---

#### Molecular Cloning

We made glycerol stocks of Dr. Jiang's strains and mini
prepped plasmids using lysostaphin. We began the process
of building recombinant plasmids using Gibson assembly. In
addition, we successfully created a BioBrick with a weak
promoter that we transformed into P. putida. We are still
working on removing illegal cut sites in RhlAB operon. We
also began the documentation and characterization process
of our parts.

#### Transformation

At this point, we have a weak promoter construct working,
yet we are still having difficulty transforming a strong
promoter in order to produce a greater degree of
rhamnolipids. We believe that this may be the result of a
metabolic burden. We brainstormed a few possible solutions
to this problem such as using conjugation methods or an
inducible promoter.

#### Rhamnolipid Quantification

This week we tried to increase the sensitivity of the CTAB
assay by using less CTAB. We discovered that the detection
limit was still too high. It was difficult to tell if the
halos were due to rhamnolipids or GFP already present in
the cell culture. Regular LB also showed halos as well,
reinforcing evidence that this assay should not be used.

TLC was utilized to quantify rhamnolipids first using a
small batch and then a larger batch of P. putida. Using
the smaller quantity of P. putida, there was too little
rhamnolipid to detect. For the larger batch, cell culture
was grown for a longer period of time and purified via
liquid-liquid extraction. Still the quantity of
rhamnolipid was too small to detect. We decided that we
should use supercritical fluid chromatography mass
spectroscopy (SFC-MS) to increase sensitivity for
measuring rhamnolipid quantity.

#### Freeze Dry

We repeated the freeze dry experiments using different
storage times: immediate and 3 days. In addition to
calculating survival rate, we also calculated the
bacterial survival ratio. The paper we were using as a
model reported an 80% "survival rate." In reality, the
survival rate they reported was a different metric called
bacterial survival ratio. This difference in terminology
explained why we thought our experiment was not
working. We had a 2.26% survival rate which corresponds to
an 82.34 bacterial survival ratio, which is the number the
paper reported. Thus, our results are on par with the
paper.

#### PhaC Knockout

We ran a PCR on the PhaC gene in knockout P. putida from
Spain against PCRed PhaC gene of WT P. putida to confirm
if a transposon was indeed in the PhaC gene. Lengths of
the PhaC gene in both strains were the same, indicating
the transposon was not in the PhaC gene.However, a PCR
product created from transposon-internal primer sequence
provided in the paper existed for the knockout but not WT,
indicating that the transposon was somewhere in the
knockout.We will do a full sequence of 200 bp upstream of
PhaC gene to 200bp downstream PhaC gene in the knockout to
get clearer picture of what is going on.

#### Mouse and Mosquito Experiments

We began the process of IRB approval and spoke with
Dr. Christiano who suggested we speak with Dr. Owens about
establishing the IRB. We also registered for lab safety
training using mice. We met with a lab member from
Dr. Owen's lab, Rong Du, to discuss the IRB in more detail
and gave her a copy of our experimental protocols. We also
improved our protocol upon discuss with a graduate
student, Lekha Nair, from Dr. Christiano;s lab.

# July

---

## 7/3-7/9

---

#### Keratinocyte Experiments

We began our first keratinocyte experiments in
Dr. Prince’s laboratory. The goal of the experiment was to
determine which MOIs of the bacteria strains (Putida,
Mutant, Arginosa) were toxic to the cells and to establish
whether the bacteria was engulfed by the cells by
plating. The experiment took longer than expected as we
included two incubation points: 2 hours and 4 hours. We
then had to leave the laboratory as there was no longer a
qualified scientist available to watch us. Before leaving,
we placed the cells on slides in preparation to count them
in a different laboratory for later use. However, by the
time we got to the other laboratory the cells had died. We
learned from this experiment that we should not make the
slides too far in advance. We also learned that we should
use a legitimate cell viability assay rather than a crude
counting method. Finally, we realized that we should split
the large experiment into smaller experiments to determine
dosage and time point this way we will have enough time to
accurately complete both experiments. Then we can use the
optimal conditions determined by these experiments in a
future culminatory experiment.

#### Rhamnolipid Production Troubleshooting

Seeing as it seems that there were no Rhamnolipids being
produced by our strain from the MS and TLC data we decided
it would be best to break down the process to a step by
step level and trouble shoot at each level. To test our
hypothesis that there was a metabolic burden, we agree it
would be best to see if a high concentration of
rhamnolipids is toxic to cell growth and test an inducible
promoter. We also thought that there may be necessary
reagents to grow such as glucose. So, we decided to
replicate the exact conditions of the primary paper from
which we were working. We questioned the promoters as
well. Should we clone the natural promoter of RhlAB into
P. Putida. Furthermore, is the bacterial strain an issue?
To answer the lattermost question, we decided to check if
transformed E.Coli produces Rhamnolipids along with
P. Aeruginosa. To analyze the transcription step we ran a
qPCR and to assess the translation step we ran an
SDS-Page.

Based upon the TLC analysis, we made the following
conclusions. First, we Confirmed that purification of
rhamnolipids via liquid-liquid extraction and TLC analysis
work efficiently for cell culture as well. Next, we
Confirmed that PAO1 mostly produces
di-rhamnolipids. Lastly, both E. Coli and P. putida do not
produce enough rhamnolipids for TLC analysis. Comparing
the MS peaks from P. Arginosa, pure rhamnolipids, and
E.Coli + RhlAB, the peak induced by our construct was not
sufficiently pronounced to conclude that rhamnolipids were
produced.

#### S. epidermidis Experiments

After adding 5-fold inserts instead of the usual 3, we
successfully PCRed out all the parts necessary to form the
two recombinant plasmids needed to perform Gibson
Assembly. Filter conjugation of empty pC221 into
S. epidermidis RP62A was successful. We made glycerol
stocks of electrocompetent S. Aureus RN4220, OS2/pGO1 and
S. Epidermidis RP62A cells. We made plans to electroplate
empty vector and construct in the coming week. To ensure
that S. epidermidis could survive the secretion of
rhamnolipids, we conducted a qualitative survival test by
directly plating S. epidermidis with the necessary 1g/L
rhamnolipids for Mosquito repellent activity. The
resultant colonies appeared more sparse in
rhamnolipid-Epidermidis cultures than cultures with Just
the bacteria. In order to determine the effect of
rhamnolipids on already established colonies, we grew
S. Epidermidis on plates overnight, and then added 1g/L
rhamnolipids to the already present colonies. A day later,
the colonies in the after one day rhamnolipid addition
plate were slightly denser than those in the immediate
addition of rhamnolipid plate, but less dense than the
plates that had absolutely no rhamnolipids present.

#### Freeze Dry

We analyzed the difference between survival rate and
survival ratio to get a better sense of the difference
between the two metrics. We conducted the experiment again
with the four day incubation. We planned future
experiments in which the incubation time would be extended
to about a month.

#### Mouse and Mosquito Experiments

Given a sample IRB from Dr. Owens we drafted our own IRB
using our protocols and sent it to Dr. Owens for review.

#### Community Outreach

Because the next generation sequencing is highly
expensive, we decided for our community outreach that this
may not be the best idea. Additionally, we would have to
undergo training for human subjects experiments and
establish a new IRB protocol. We still thought it was
beneficial to develop a survey and FAQ about mosquito
borne illnesses. We drafted a version of the survey asking
questions about frequency of mosquito bites, knowledge of
mosquito borne illnesses, and preferred characteristics of
mosquito repellent (ex: is scent important when choosing a
repellent?). We sent the survey to a cohort of individuals
as a pilot study to find any errors in the survey data and
gain preliminary results. The power analysis revealed that
we need about 250 participants or more to obtain
statistically significant results.

## 7/10-7/16

---

#### Rhamnolipid Quantification

We grew wild type P. Aeruginosa and transformed
P. Aeruginosa with L1 + RhlAB, and extracted rhamnolipids
from the two strains using liquid-liquid extraction.
SFC-MS result shows that mutant P.A. produces much more
mono-rhamnolipids, confirming that our construct works
well in P. Aeruginosa. We also tried growing mutant
Putida and E. Coli (transformed with L1 + RhlAB and H2 +
RhlAB) in a different growth media, LB + glucose. SFC-MS
result shows that all of them make mono-rhamnolipids:
E. Coli + H2\_RhlAB made the most mono-rhamnolipids, then
E. Coli + L1\_RhlA, then putida + L1\_RhlAB.

#### Transformation of P. putida PhaC knockout

We tried transforming the knockout strain with 4 different
promoters and RhlAB, but only L1+RhlAB showed GFP positive
colonies. We will sequence it to confirm the operon and
quantify the amount of rhamnolipids.

#### Protein Analysis

We ran the gel, but it was unclear and blurred because we
did not purify the gel. However, there were bands of the
right size for RhlA and RhlB. MS showed that rhamnolipids
were detected. So, we decided not to spend the time
purifying the gel.

#### qPCR

Results show that mRNA is being properly synthesized by
our construct, although our negative controls (wild-type
and empty vector) indicated some unexpected mRNA.

#### Keratinocyte Experiments

We obtained our cells, but they were highly confluent
during the weekend. We performed experiment testing effect
of different bacterial strains (paK, WT PP, PP+L1+Rham,
and WT PP with 1g/L mixed rhamnolipid) with different MOIs
(10, 100, 1,000, 10,000, 100,000) on keratinocyte
viability. Cell survival was testing with the MTS cell
viability assay, which measures the level of live cells
per sample. Keratinocytes were first plated at 3x10^4 per
well into 48 well plates, then weaned off P/S antibiotics
on the next day. The following day, the different strains
were each added with the different MOIs with 3 trials per
situation. Positive control was a "0 MOI" situation with
no bacteria added, which should represent maximum cell
viability, and two negative controls were pure media and
pure media with 100,000 MOI of each bacterial strain,
which should give a cell viability of 0. This was repeated
at 2 time points: 3 and 24 hours. Data from the 3 hour
time point has been collected.

#### Molecular Cloning

We tested the Staphylococcus protocols for miniprepping,
electroporation, and filter-mating with empty vectors. We
also constructed our recombinant pC221+RhlAB+GFP and
pC194+RhlAB+GFP plasmids for transformation into the Staph
strains. To more closely observe the effect of
Rhamnolipids on S. epidermidis, we performed a gradient
plate experiment which once again showed sparser colonies
for S. epidermidis with higher concentrations of
Rhamnolipids. We decided that designing a quantitative
experiment with a 96-well plate, instead of just relying
on qualitative experimentation was necessary.

## 7/17-7/23

#### Metabolics Troubleshooting

Given the low yield of rhamnolipids from the successfully
cloned promoters of our promoter library, alternative
promoters were explored to possibly boost production.
Experiments were planned to more closely replicate the
Wittgen paper by extracting the RhlAB operon with its
natural promoter by PCRing out the operon along with 200
extra bps upstream. Cloning this new operon had limited
success and rather than repeating experiments it was
deemed less relevant. More priority has been placed on
designing/extracting the Xylose inducible promoter given
that it might be viable for usage in Staph epidermidis as
well.

#### Promoter Characterization

Most of the higher strength promoters have been unreliable
in terms of transformation and rhamnolipid production. It
is speculated that the issue with this lies in that they
are constitutively triggering the RhlA gene to make the
rhamnolipid precursor HAA while the RhlB is only active
during the stationary phase. Buildup of HAA might be
causing the bacteria to view the operon as impeding
survivability and in some way shut it down. This could be
solved using the Xylose inducible promoters so that the
buildup of HAA is prevented and high levels of
rhamnolipids would be produced upon induction in the
stationary phase.

#### Keratinocyte Experiments

Keratinocytes were treated with different MOIs of bacteria
strains Putida, Arginosa, Mutant, and Putida with
Rhamnolipid to determine the relationship between
bacterial concentration and cell viability. The cells were
treated with gentamicin after 3 or 24 hours and then an
MTS assay was utilized to quantify cell viability. The
cells were then plated to deduce if bacteria had been
drawn in by the cells. The results showed that there was
not a significant difference in the bacterial
concentrations, which may indicate that the serial
dilutions were incorrect. Additionally, there was a high
degree of bacterial growth on the plates, which was not
expected. Perhaps, the gentamicin did not kill the
bacteria on the cells either because the concentration was
too low or the the incubation time period was not long
enough.

Another experiment was conducted to determine the IC50 of
rhamnolipid by treating the keratinocytes with different
concentrations of rhamnolipids for 24 hours. The
concentrations selected were based upon the results of
several research papers that utilized rhamnolipids with
different cell lines. Graphpad software was utilized to
model the the data and it was determined that the IC50 is
between 40 and 60 ug/ml.

## 7/24-7/30

#### Molecular Cloning

Tired of waiting for the xylose inducible promoter
backbone plasmid, we decided to utilize what we have
already available in the lab and clone arabinose inducible
promoter into our construct. We designed and ordered the
primers for the new construct with the arabinose promoter.

#### Rhamnolipid Quantification

This week, we wanted to see if saturating LB with glucose
helps producing rhamnolipids. We grew mutant putida with
LB + 10g/L of glucose, 30 g/L of glucose, and 50 g/L of
glucose and measured the amount of rhamnolipids at
different time points. After 24 hours of growth, 50 g/L
glucose gave the most amount of rhamnolipids. After 72
hours of growth, all three growth media gave slightly less
rhamnolipids. It is possible that bacteria are eating up
rhamnolipids as they run out of carbon sources during the
stationary phase. We also created a more accurate standard
curve using 95% pure mono-rhamnolipids.

#### Keratinocyte Experiments

Based upon our suspicion that the gentamicin was not
killing the bacteria, we conducted an experiment in which
we manipulated the concentration of gentamicin and the
incubation time points using four different strains of
bacteria (E.coli, W.T. Putida, Aeruginosa, and Mutant
strain). The highest time point and concentration was four
hours at 200 ug/mL yet these conditions were not
successful on all of the strains.

We planned repeat the bacteria MOI experiment with
different, accurately calculated MOIs knowing the new
information about gentamicin incubation time and
concentration. Because the 4 hour time point was
unsuccessful, we were unsure for how long to incubate the
cells in gentamicin before completing the MTS assay. Thus,
we decided to leave the cells in gentamicin overnight and
concomitantly treat a separate bacterial culture with
gentamicin overnight to compare the bacteria growth.

We encountered another problem with the experiment because
our control cells were contaminated by bacteria. We could
not bring the bacteria into the cell culture hood so we
brought the cells to the bench when we added the bacteria
under flame. Even with these precautions there was
contamination. After discussion, we decided to continue
the experiment by treating with gentamicin overnight,
thinking that the gentamicin will kill the contamination
in the negative control conditions as well. While this
experiment may not yield all the results we wanted
(specifically bacteria intake by the cells), we will be
able to determine if there is a difference in cell
viability given different MOIs.

#### Transformation of S. epidermidis

We successfully transformed pC194\_RhlAB into the shuttle
vector for electroporation, S. Aureus RN4220, however
transformation of pC221\_RhlAB into the conjugation vector,
OS2/pGO1, was unsuccessful. We re-tried electroporating
pC221\_RhlAB into OS2/pGO1 a couple more times, but we were
unsuccessful. We also conducted the 96-well S. EPidermidis
Experiment, however, the Rhamnolipid blank began to grow
around the 7;12 hr mark, making it necessary for us to
disregard the results.

## 7/31-8/6

---

#### Metabolics Troubleshooting

We have successfully cloned the arabinose inducible RhlAB
backbone and transformed into 10-beta E. Coli strain. We
have also transformed it into Putida, which gave us
bacterial lawns. Surprisingly, the lawns were GFP
positive, and we learned that Arabinose isn't really
inducible in Pseudomonas. We will still compare its
expression level to our original construct.

#### Keratinocyte Experiments

We conducted more experiments to determine at what
concentration of gentamicin and time point successfully
kills the bacterial strains. However, they were
unsuccessful again. We spoke to our mentors and decided
that a biofilm may be forming that prevents the bacteria
from dying. We conducted OD experiments to determine at
what point the bacterial strains are in mid exponential
phase. The results were inconclusive for all of the
bacterial strains except for E.Coli. Thus, we tried to
conduct an experiment to assess which MOIs of E.Coli cause
cell death but there was an error with our cell culture
and the cells were dead in the morning. We now think that
the problem with our experiments is that we are using very
high bacteria concentrations. We made plans to repeat the
experiments with lower concentrations.

#### Molecular Cloning

We successfully electroporated pC221\_RhlAB into S. Aureus
RN4220. We intend to electroporate mini-prepped pGO1
helper plasmid into RN4220, and then filter conjugate from
here, since we were unable to electroporate pC221\_RhlAB
directly into OS2 which already contained pGO1. After
Millipore filtering our Rhamnolipids solution, we repeated
the 96-well S. Epidermidis vs. Rhamnolipids survival
test. Our negative control was S. epidermidis with 2uL of
34 ng/uL Kanamycin, while our positive control was
S. epidermidis with just BHI. After adding a 1/1000th
dilution of overnight to a range of two-fold serial
dilutions from 2000mg/L to 15.6mg/L. The resultant OD600s
showed that growth of S. Epidermidis is staggered by
higher concentrations of Rhamnolipids up to125mg/L of
Rhamnolipids, although the OD600 Does eventually get to OD
1 by the 12th hour. So although production of
Rhamnolipids in the high concentrations we need might
stagger the growth of S. epidermidis, it will not
necessarily kill our cells.

#### IRB Update

We have submitted a draft of IRB amendments to IACUC,
which we are adding on to Dr. Owens protocol. Since we are
changing Dr. Owens’ pain level to D, as a result of the
skin-grafting component of our mouse experiments, we are
getting a vet approval which will be sent to the IACUC as
well this week. We have also been assigned an IACUC
consultant, Sierra Fuller, who has been advising us on the
level of detail that must be included in the protocol.

#### Marketing

We have started thinking about how we can market our
mosquito repellent. Using AutoCad, we will 3D print our
marketing bottle!

# August

---

## 8/7-8/13

---

#### Metabolics Troubleshooting

It looks like there was a recombining when we transformed
the arabinose inducible promoter plasmid into Putida.
Even though the colonies were GFP positive, sequencing
result gave us a negative result.

#### Keratinocyte Experiments

This week the cells died because they were contaminated
with bacteria. We plated the cells to confirm our
hypothesis. We contacted the laboratory that gave us the
cells. The graduate student will not have them available
for another few weeks because their cells were also
contaminated. Thus, these experiments will be put on
hold. However, we decided to try a different assay
technique in which we lyse the cells and then measure
bacteria presence. This will avoid the complications
regarding the concentration and duration of gent
application to the cells.

#### Community Outreach

We created a map based upon the survey data illustrating
what regions of the country experience more mosquito bites
than others. We collaborated with the University of
Georgia and assisted them on their project. We also
contacted BioBus an organization in which scientists teach
students about the field while they are on a bus to plan
an event with them. However, the scientists will not be
back to work until September. We made arrangements to
contact them in September. Additionally, we made contact
with the Chinese iGEM Team who is putting together a
newsletter and asked if we would like to contribute.

## 8/14-8/20

---

#### Mosquito Experiments

Last Friday, we sourced Aedes Aegypti mosquitoes from the
Vosshall lab at Rockefeller University, who kindly gave us
two large tupperwares full of larvae and pupae and braved
NYC traffic to drive us back to CUMC. The mosquito life
cycle consists of 4 stages: eggs, larvae, pupae, and
adult. On Saturday, Sunday, Monday, and Tuesday, we sorted
out the male mosquitos (cannot suck blood) in both pupal
and adult stage. The pupae are sexed by size (females are
larger) and adults are sexed by antenna (males have fuzzy
antennae while females do not)[insert picture] We spend
countless hours in the cold room sorting adult and pupal
mosquitoes because the cold prevents mosquitoes from
escaping.

#### Keratinocyte Experiments

This week we conducted an experiment using smaller
bacterial MOIs to determine if the high MOI was the reason
that gentamicin was ineffectual. All four bacterial
strains were plated at MOIs that do not exceed 10. The
following day there was no evidence of bacterial growth!
Thus, when the new keratinocytes are received this
concentration of gentamicin can be utilized in the
experimental procedure.

#### Community Outreach

We arranged an event scheduled for October 1st with the
organization ThinkSTEAM that promotes science and math
education in girls. The program will consist of a
presentation introducing systems biology, research
overview, and laboratory demonstrations.

## 8/21-8/27

---

#### Keratinocyte Experiments

This week we obtained the flask from the Prince Lab
because they resolved the microplasma problem. I fed the
flasks and split them. However, when I came back to the
lab after the weekend there were fungi growing in the
flasks. I checked the incubators and there is a warning
that filters need to be replaced, which may explain the
contamination. I notified the laboratory of the
contamination issue. I contacted other laboratories in the
building to see if we can use incubators while waiting for
the incubators to be replaced and one laboratory agreed.

## 8/28-9/3

---

I was able to plate cells this week and conduct an
experiment to determine if E.Coli causes cell death. We
utilized a new essay from a paper entitled,
"Internalization of Pseudomonas aeruginosa Strain PAO1
into Epithelial Cells Is Promoted by Interaction of a T6SS
Effector with the Microtubule Network." We utilized the
gent concentration specified in the article and lysed the
cells prior to plating. The experiments revealed that the
bacteria did not cause significant cell death at any
concentration. Additionally, when the cells were plated
bacterial colonies did not grow, indicating that the
bacteria was not internalized by the cells.

116th Street and Broadway, New York, NY 10027

[Safety](https://2016.igem.org/Team%3AColumbiaU_NYC/Safety)

[![Twitter](https://static.igem.org/mediawiki/2016/e/e4/Twitter_Logo_Blue.svg)](https://twitter.com/columbia_igem)
[![Facebook](https://static.igem.org/mediawiki/2016/e/e8/FB-f-Logo_blue_1024.png)](https://www.facebook.com/columbiaigem/)

[![](https://static.igem.org/mediawiki/2016/b/bf/HQ_menu_logo.jpg)](https://2016.igem.org)