iGEM 2018 — Team TecCEM

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# Team:TecCEM/Notebook

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* [Home](https://2018.igem.org/Team%3ATecCEM)
* Project

  [Description](https://2018.igem.org/Team%3ATecCEM/Description)
  [Design](https://2018.igem.org/Team%3ATecCEM/Design)
  [Results](https://2018.igem.org/Team%3ATecCEM/Results)
  [Experiments](https://2018.igem.org/Team%3ATecCEM/Experiments)
  [Hardware](https://2018.igem.org/Team%3ATecCEM/Hardware)
  [Software](https://2018.igem.org/Team%3ATecCEM/Software)
  [InterLab](https://2018.igem.org/Team%3ATecCEM/InterLab)
  [Model](https://2018.igem.org/Team%3ATecCEM/Model)
  [Demonstrate](https://2018.igem.org/Team%3ATecCEM/Demonstrate)
  [Improve](https://2018.igem.org/Team%3ATecCEM/Improve)
* Parts

  [Parts](https://2018.igem.org/Team%3ATecCEM/Parts)
  [Basic Part](https://2018.igem.org/Team%3ATecCEM/Basic_Part)
  [Composite
  Part](https://2018.igem.org/Team%3ATecCEM/Composite_Part)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3ATecCEM/Human_Practices)
  [Public
  Engagement](https://2018.igem.org/Team%3ATecCEM/Public_Engagement)
* [Notebook](https://2018.igem.org/Team%3ATecCEM/Notebook)
* Team

  [Team Members](https://2018.igem.org/Team%3ATecCEM/Team)
  [Collaborations](https://2018.igem.org/Team%3ATecCEM/Collaborations)
  [Attributions](https://2018.igem.org/Team%3ATecCEM/Attributions)
* [Safety](https://2018.igem.org/Team%3ATecCEM/Safety)
* Awards

  [Hardware](https://2018.igem.org/Team%3ATecCEM/Hardware)
  [Software](https://2018.igem.org/Team%3ATecCEM/Software)
  [Measurement](https://2018.igem.org/Team%3ATecCEM/Measurement)
  [Model](https://2018.igem.org/Team%3ATecCEM/Model)

![Cell Gif](https://static.igem.org/mediawiki/2018/e/ef/T--TecCEM--Cells--Notebook.gif)

# Notebook

### Project/ Notebook

[Index](#notebookSubmenu)

* Week 18th
* Week 19th
* Week 20th
* Week 21th
* Week 22th
* Week 23th
* Week 24th
* Week 25th
* Week 26th
* Week 27th
* Week 28th
* Week 29th
* Week 30th
* Week 31th
* Week 32th
* Week 33th
* Week 34th
* Week 35th
* Week 36th
* Week 37th
* Week 38th

##### Week 18th **13 - 19 May.**

## Summary

Research for characterization began and sub-projects deadlines were assigned.
Logistics of the Latin American Meet-up began. A visit to Six Flags was made.

### Wet lab

Preparation of solutions to be used for the competent cells. Transformation
efficiency for E. coli DH5α and antibiotic viability were tested.

### Dry lab

Delimitations of the mathematical model were made. Pitching of software ideas
and sub-projects. We started looking into the possibility of creating a
skinchip in a mini reactor. Investigation about the scaffold heterotrimer was
appointed. The development of biobricks for recombinant leptin and collagen was
started. A meeting with an expert in miRNA therapy was appointed, new insights
about anti-miRNA were established from that meeting and the viability was
researched. Leptin and collagen were codon optimized for their recombinant
production in Escherichia coli BL21 (DE3) and Rosetta (DE3) respectively.

### Human Practices

Committee for human practices was delimited. A brainstorming of ideas was done
and the best ideas were picked up in order to develop them in the near future.

##### Week 19th **20 - 26 May.**

## Summary

Worklab began and we had meeting to assign new sub-project. Wiki page
planification began, we contacted a Graphic designer to help out. We also
contacted several specialists in tissue regeneration to gain insights about the
project.

### Wet lab

Lab got closed off for maintenance reasons. The iGEM kit got stuck at the
customs border protection.

### Dry lab

Skinchip 3D model was designed and software ideas were pitched. Biobricks were
checked and revised. Chemical modifications for anti-miRNA were investigated,
locked nucleic acid (LNA) technology appointed as the best method for miRNA
silencing. Receptors within the scaffold for stem cell migration/call was
investigated but also other molecules apart from the main project
protein/molecules were investigated for the same purpose.

### Human Practices

Fundraiser organization and planification, tasks were handed out. Went to a
public talk about tissue regeneration in burn victims using nanoparticles of
chitosan, we made contact with the people involved in the conference and
received a feedback, another meeting with the experts was appointed. We had a
meeting with a communication reporter to be published in a magazine. Online
meet up with iGEM Brazil, Ecuador and Chile were performed.

##### Week 20th **27 May - 2 June.**

## Summary

Worklab began and we had meeting to assign new sub-project. Wiki page
planification began, we contacted a Graphic designer to help out. We also
contacted several specialists in tissue regeneration to gain insights about the
project.

### Wet lab

Transformation of competent E. coli DH5 cells was performed using different
plasmids, no successful transformation was seen. New cells were created under
the same protocol. Escherichia coli DH5 transformed with psB1C3 and inoculated
in LB medium. A Skin chip prototype was manufactured using PDMS and normal
objects such as clips and shelf sustainers.

### Dry lab

Protein sequences for the ECM were optimized with IDT and verified. Novedous
methods for the miRNA silencing were analyzed theoretically and several
critical factors for the competitive binding against RISC complex were
encounter. Physico-chemical characteristics for leptin were revised and
documented. Several designs were proposed for the modelling and manufacturing
of the skin chip.

##### Week 21st **3 June- 9 june.**

## Summary

We had a meeting with Doctor Pablo Rodriguez from the Special Center for burn
children of the General Hospital of Toluca Dr. Nicolás San Juan.

### Wet lab

We transformed E.coli DH5 alpha with non important plasmids, in order to verify
efficiency. Also alkaline plasmid extraction were done tested with
electrophoresis.

### Dry lab

Biobricks were revised. Chitosan and leptin union to scaffold protocol was
designed. We sent our sequences to IDT for synthesis. Skinchip design was
created and defined an operational model.

##### Week 22nd **10 June- 16 june.**

## Summary

This week we recorded our video for a fundraiser. The Latin American Meetup
logistic is being planned and organized. We had pizza on friday.

### Wet lab

We re-tested alkaline plasmid extraction protocols and reviewed it with
electrophoresis. Cell culture area was sterilized for HaCAT cell line
subculture. Antimicrobial peptide LL37 from Trieste team contained in iGEM
Plate Distribution 2017 was transformed in DH5-alpha.

### Dry lab

Chitosan protocol revised and changed after a meeting with an expert. iGEM’s
kit is still stuck at the customs border. The docking between proteins was
modeled. A skinchip model was designed and revised with several experts.

### Math model

The math model for pharmac diffusion along a matrix was investigated, the
equation of Weilbull was chosen and variables for leptin were investigated.

##### Week 23rd **17 June- 23 june.**

## Summary

This week a lot of progress was made, we had a meeting with Doctor Gerardo
Manuel García Lozano of Foundation RinoQ hospital. We had courses that dived
into tissue and cell culture and the correct lab safety cautions that need to
be taken from Laboratorios de Especialidades Inmunológicas SA de CV (LEI), a
laboratory that specializes in immunology and tissue cultures. Our Interlab Kit
was greenlighted by the customs border!

### Wet lab

HaCAT subculture was made. L929 cell line was established and given the correct
maintenance and subcultures were made. Primers for VF2 and VR were obtained and
Agilent Assembly Kit primers designed and. We dried plasmids, in order to send
it to iGEM team TecChihuahua. A PCR using iGEM consensus for LL37 was
performed.

### Dry lab

The skinchip was modeled in 3D and the bioreactor workflow chart created.
Research is being revised and protocols created for lab work.

### Human practice

Several arrangements were performed with RinoQ foundation to coordinate the trip
to visit childrens that have suffered of burns and they helped us by giving
feedback about the project. A visit with Fernando Osobampo, a specialist in
plastic surgery, that has had a lot of close contact with the recovery of
people that had suffered of burns, we had a lot of feedback and focused our
project in second degree burns and had an approval of the efficiency of our
delivery method.

##### Week 24th **24 June- 30 june.**

## Summary

This weekend we continue with the realization of the characterization of LL37.
Several advances were created in order to organize the first main human
practice of the team. Hardware and mathematical models were finally design. A
visit with a specialist in plastic surgery was realized. The iGEM distribution
kit arrived at our laboratory (26/07/18).

### Wet lab

The primers for Agilent kit were resuspended in order to create workable stocks
(10 M). A enzyme restriction was performed to analyze the LL37 sequence
amplified last week. The Agilent kit reactants were aliquot into workable
stocks as the protocol indicated. Chitosan first nanoencapsulation was
performed using a negative control (water) and a lipase for encapsulation.

### Dry lab

Agilent Sure Vector kit protocol was read.

##### Week 25th **1 July- 7 July.**

## Summary

On this weekend the laboratory was mostly closed because of maintenance and the
visit to RinoQ was done. The characterization of LL37 continued by making the
first step to the assembly protocol for Agilent kit.

### Wet lab

A PCR using the agilent synthesized primers was performed, in order to attach
the necessary sequences for the overlap used in the assembly of the plasmid.

### Human practice

A visit to RinoQ foundation, in San Luis Potosí, was achieved. An assessorament
with experts in the burns area was done to make the project more efficient. A
coordination between TecCEM and RinoQ was created, in order to create an
application to help burn victims and to prevent burns.

##### Week 26th **8 July- 14 July.**

## Summary

We had the iGEM Latin America 2018 Meet Up! We had the pleasure to receive teams
from all over Mexico and Latin America such as Brazil, Ecuador, and Chile where
they presented their projects and got feedback from specialists. This Meetup
was important for the teams to get an overall look into their work and
something to note is that the event got a lot of diffusion online.
We also started the planification and logistics for Universium as next week the
event would take place.

### Wet lab

The first try to do an assembly of LL37 was performed using the previous LL37
fragment and a PCR cycle with all the reactants of the Agilent kit.
Successfully after the second try the plasmid was assembled with the LL37
sequence, this was done by increasing the number of cycles to 16. This assemble
gave us a plasmid that contain a T7 promoter, bacterial Ori, Amp resistance,
terminator and the LL37 protein along with a His tag.

### Human practice

The iGEM Latin America Meet Up had place in our campus, we had the presence of
teams from Monterrey, Guadalajara, Chihuahua and virtual presence of teams from
Brazil, Chile and Ecuador. In this meet up the teams had the opportunity to
give a presentation such as pre-run to practice the one that has to be done in
Boston, in addition several specialists were there to analyse and give feedback
in the projects. The logistics of the visit to Universium was performed.

##### Week 27th **15 July- 21 July.**

## Summary

We had meetings with several teams seeking if we could collaborate with them and
see how the rest of the iGEM teams are doing to try and expand communication
around the scientific community. We also went on with the preparations for
Universum. We continued with the characterization of LL37 and the
transformation of E. coli. The DNA synthesize from IDT was delivered and the
first project insights could be achieved. This week Interlab was done in the
lab to obtain the results.

### Wet lab

The plasmid with LL37 was transformed in E. coli strains as Rosetta gami and
BL21 (DE3), the transformations were successful as single colonies were
achieved. In order to verify the transformation plasmid extraction was
performed and positive results were obtained. The IDT sequences for collagen
type V (COL5), tenascin domain V (TCD5) and leptin (LEP) were resuspended in
bio mol water for the creation of stocks. A ligation to the plasmid psB1C3 was
performed by enzymatic restriction and T4 DNA ligation.

### Human practice

The visit to Universum was done in the weekend, during this time we explained
biotechnology concepts to children and gave them a little insight to the
subject. We also talked with their parents to explain more about the science
field in genetic engineering and the project in general terms, by doing this we
expected that biotechnology and the general public can come into contact and
become a subject of interest for everyone.
In addition, we took this opportunity as a way to gather some data by doing a
survey, which will help us in the development of an app for burn prevention.

##### Week 28th **22 July- 28 July.**

## Summary

This week we finished Interlab and sent the forms to iGEM, we continued as well
to communicate with other iGEM teams from around the world to collaborate and
we started to organize with them to do the video. We advanced with the
transformation of the iGEM parts in E. coli strains and with the design of our
hardware, but also we started seeking for the establishment of an experiment
which could asses our project viability.

### Dry lab

In order to evaluate our project we started the searching of a protocol which we
could use in our human cell lines to recreate the effects caused by burn
accidents with either hot liquids or hot surfaces, which was told by the
foundation Rhino Q to be the most common ones. We obtained two main articles
which had a lot of potential to our project, both articles described the
methodology in which we could treat our cell lines to recreate this burning
effect and also it was described how we could measure it. By those means our
assays would involved the usage of a hot glass Petri dish and the evaluation of
cell viability by the usage of MTT or other molecules.

### Wet lab

We transformed E. coli DH5 alpha for plasmid multiplication purposes resulting
into correct results for BBLEP and BBTNC5, for BBCOL5 the observance of red
colonies were determined as a non conclusive result because of the problem that
BBCOL5 part construction had a constitutive RFP as a reporter but the
re-ligation of any iGEM plasmid also results in a RFP but controlled by a lac
promoter, in order to verify the results obtained a posterious induction / non
induction experiment must be done to determine the veracity of the ligation. We
made a quick trial into the recreation of burn assay into our cellular lines,
we obtained interesting results showing that the exposure of 15, 30 and 60
seconds to PBS at approximately 80oC showed a decrease in cell morphology and
cell viability, the results were obtained by cell counting in Neubauer chamber
with trypan blue and microscope visualization.

### Hardware

We came to a possible proposal of a skin chip and designed a 3D model including
the mechanisms, the proper calculations and how to automate the system. This
model still very naive and is needed to get proper correction in order to
create the first design of the skin chip.

### Interlab

The parts of iGEM kit plate distribution was transformed in E. coli DH5 alpha
strain and alkaline plasmid extraction was performed and agarose gel analysis
was done to verify plasmid existence. Calibration and cell measurement
protocols were performed and submitted in the wiki
(https://2018.igem.org/Team:TecCEM/InterLab).

##### Week 29th **29 July- 4 August.**

## Summary

This week was the last week of summer break so we had to work harder than ever.
A briefing about what was done during the summer was broken down in a meeting
and a new planification of objectives was done.

### Wet lab

We performed the transformation of all project parts (BBCOL5, BBLEP and BBTNC5)
into protein production strains such as BL21(DE3) and Rosetta (DE3). After
alkaline plasmid extraction and analysis in electrophoresis we could conclude
that the existence of the parts project BBLEP and BBTNC5 were achieved, even
though several more analysis must be performed in order to obtain significant
results. In the case of BBCOL5 an experiment of induction/non induction was
performed to analyse if the ligation product was a re-ligation of iGEM plasmid,
the results obtained concluded that the part was indeed a re- ligation product
and for so on BBCOL5 ligation was discarded as part of the project but used as
a medium to obtaining RFP for nanoencapsulation experiments.

### Hardware

We contacted COMSOL for their software specialized in microfluidics also a
professor in our campus that specializes in microfluidic modelling was
contacted for tutoring.

##### Week 30th **5 August- 11 August.**

## Summary

This weekend we started classes in our university, also we had significant
advances in the verification of plasmid existence (project parts) in bacteria
colonies. We started production and purification of protein of interest. We had
an incident in the lab too, our centrifuge broke and the cellular lines died!

### Dry lab

We tried to make a custom medium broth for collagen production and consulted
with a professor to reassure the calculous sheet was correct but the idea was
shut down because it could have had too many variables.

### Wet lab

We started with protein production of protein LL37 of E. coli strains: Rosetta,
DH5a and BL21. The electrophoresis analysis in polyacrylamide gel of LL37
protein production was performed but no results were achieved, this could have
been due to lysis protocol, thus new purification protocols were revised. The
cellular lines of HaCaT and L929 had died because of a runout accident of CO2
master tank, producing acidification and loss of morphology, plans for
obtaining new aliquots of cellular lines started. The device BBCOL5 was tried
to ligate into plasmid pSB1A3, but no consistent results were achieved.

##### Week 31st **12 August- 18 August.**

## Summary

This week we had several team meetings to get some team building going and
continued the protein production for characterization. Also the first advances
of a complete hardware were finally achieved.

### Wet lab

New protocols for purification of LL37 protein were done. RIPA buffer lysis
protocol was used but polyacrylamide gel analysis was not achieved because of
difficulties in visualization. We improved our polyacrylamide protocol and got
a new Coomassie solution given as a gift from one professor which really helped
us to visualize our gels more easily. For protein production analysis of the
project parts (BBLEP and BBTNC5), an induction curve was performed, in which
samples of each device transformed into BL21(DE3) were analyzed in a time lapse
of 9 hours after IPTG induction, taking aliquots of 1 mL per hour for
posterious protein analysis experiments, each aliquot was done by duplicated
and observed in spectrophotometer at OD600 to correlate protein production with
total biomass. Also we obtained new HaCaT cell lines.

### Math model

We had a meeting with several professors to help us get a better idea about the
whole math model and the reach our project could have.

### Hardware

Models for skin chip were design using AutoCad and Catia softwares, the
modelations of flows were done and the materials needed were investigated.

##### Week 32nd **19 August- 25 August.**

## Summary

This week we had several advances into the software and hardware part of the
project. The protein curve production was analysed by polyacrylamide gel. The
first insights into RFP purification were obtained.

### Wet lab

The protein curve was analyzed by polyacrylamide gel and the results obtained
were not good enough, several factors affected the quality of the gel. We
started with nano encapsulation of RFP and visualization of nanoparticles in
SEM microscoped.

##### Week 33rd **26 August- 1 September.**

## Summary

Our protein production began, we consulted a professor specialized on proteins
that gave us several tips on it. We reworked the hardware design as a final one
that is less expensive and more innovative, then we sent the design into a
company so the corp was made.

### Wet lab

We created a new production protein curve and performed an analysis by
electrophoresis and Bradford quantification. The polyacrylamide gel was done at
20% to obtain a better separation of protein, although no significant
overproduction bands were seen. The quantification by Bradford methodology
showed a low quantity of protein in pellet centrifugation. This results made us
re-evaluate the lysis protocol and change the quantification methodology. We
made contact with a professor expert in recombinant protein production and we
obtained new crucial points for a new definitive curve. We made nano
encapsulation with RFP and saw it in SEM microscope, no structure was seen, we
started seeking for new methodologies for obtaining better resolution in the
results. We performed a new assay of in vitro skin burn in L-929 cell line.

### Hardware

The manufacturing of the skin chip was investigated, the final material used for
this purpose was glass as it can be reused and can help labs to cut their
dependence in trash-able cell culture bottles. Several companies were contacted
in order to quote prices, and at the end the company “” was contacted to create
the skin chip carcass.

##### Week 34th **2 September - 8 September.**

## Summary

On this week we retried the curve of protein production of BBLEP and BBTNC but
applying the points that our professor gave us, the results obtained made us
replan the project methodology into a fresh beginning.

### Wet lab

After the results obtained last week we set the objective to prove that our
proteins could be produced by the bacteria. For this to be achieved we made a
new production protein curve with different new settings such as: BL21(DE3)
with another plasmid to corroborate that bands seen were not result of
antibiotic resistance peptides, creation of a non induced protein curve, and
starting induction until OD600 of 0.7. The results obtained were not
significant and there were no bands showing our proteins. Also we performed a
PCR to an alkaline extraction of plasmids coming from BL21(DE3) transformed
strains (BBLEP and BBTNC), using VR and VF2 primers, the results obtained show
no amplification of BBLEP and inconsistent results for BBTNC. With that on mind
we aimed to redo the transformation of parts and protein production, in order
to obtain new and much better results. We also obtained kanamycin for the
ligation of BBCOL into psB1K3. We realized the quantification of lactate of our
burned cell lines at 0.5, 1, 2 and 3 minutes, in order to get numerical data
that will translate into proliferation rate after a burn accident, those
results will help us into developing a more accurate math model for the
purposes of the project.

### Dry Lab

We started to investigate new lysis protocol to ensure that our protein was
released from the bacterial pellet, we obtained one protocol which was specific
for the purification of tag His proteins derived from BL21(DE3), but also we
attended with our professor specialist in protein purification for new
protocols, she gave us a Laemmli buffer for fast visualization of results for
protein induction. Also we received the idea from one of our assessors to use
RIPA buffer for the lysis, but after an extensive internet research no
methodology was seemed to work with E. coli pellets.

### Hardware

We went to CDMX to obtain the hoses and syringes for the assembly of the skin
chip. Also we talked with professor Chong to land the idea of how we will be
measuring the proliferation rate in the skin chip, the idea ended up in the
usage of Tsc3200, which is a programmable converter of light (colour) to raw
data, that will measure the virage (acidification) of DMEM medium from rose
into orange, which is due to the metabolism of cells and would be a good
parameter for account how much the cell is growing given the application of the
project and when those the system must change the medium.

##### Week 35th **9 September - 15 September.**

## Summary

This week we re-organized the project objectives according to a our new
chronogram and replanificated our biggest goals to achieve the production and
purification of our proteins, also nanoparticles creation was performed. We
started our production from ground zero as we were not having any positive
results, and as a way to troubleshoot this we made new competent cells to
produce our proteins. Nonetheless, the hardware prototype was finally obtained
and some tests were performed to get insight into it’s functionality.

### Wet lab

The production and purification of RFP was successful by implementing the new
lysis buffer, even though, a modification in the buffer must be performed to
increase the lysis efficiency given that there was still red color in the
pellet. Using the resultant RFP we started the nano encapsulation, the result
was store at 4oC. We started the creation of completely new BL21 (DE3)
competent cells for the posterious transformations of newly ligated parts. We
obtained kanamycin for the implementation of psB1K3 in the ligation of BBCOL5
part. We performed the transformation of the new BL21 (DE3) chemocompetent
cells and we obtained white colonies, by that mean, we performed induction of
protein expression in the new colonies to completely verify the presence of our
proteins, for a posterious polyacrylamide gel analysis. Sadly, after the
incomplete subculture of L929 given by the loss of functionality of our
trypsin, no good cells could be incorporated into the new flask and because of
that we lost our cell line, for so on we must obtain another aliquot.

### Dry Lab

We researched a new lysis protocol for purification of proteins. The protocol we
obtained was specific for cell lysis and nickel affinity purification of E.
coli BL21. From that protocol we keep special attention in the lysis buffer
which is comprised of many reductant agents. Also in an article which described
the lysis method for E. coli we found that his lysis buffer was exactly the
same as the one we previously encounter, for that we decided to give that
buffer a try but with modifications according to our experience such as
incubation with lysozyme.

### Hardware

We obtained the prototype of the “skin reactor” and performed a test to
determine exhaust capacity of the prototype using only inclination and
centrifugal force. The results obtained showed that an angle of 7.5° was
necessary to remove most of the medium from the medium chamber. For that the
creation of a base would be necessary for the removal of medium given those
characteristics. We then proceed to modelate the base in CATIA and later we 3D
printed it in our school.

##### Week 36th **16 September - 22 September.**

## Summary

Great news ! On this week we started gazing the first possible obtention of our
proteins. Also we created more important components for the transformation of
BBCOL5. The hardware was improved by incorporating all the components necessary
for his functionality.

### Wet lab

For the nanoparticles we then went to IPN institute to use their TEM
(transmission electron microscope) and NanoSight to show the results obtained,
we could proudly say that the first extraction of RFP nanoparticles was
achieved and a good image from TEM was obtained.

### Hardware

The final assemblies were done and the automatization parts were laser cut for
their incorporation.

##### Week 37th **23 September - 29 September.**

## Summary

This week we began working way harder in the lab as deadlines approach. We
decided as a team the wiki order and design, as well as a name for our
hardware.

### Wet lab

Nanoparticles standardisation was done with Bradford assay for a quick
encapsulation once Leptin begins production. PBS pH 7.4 was used to recreate
physiological medium and test nanoparticles liberation.

### Hardware

The last assemblies were done and the sensors were tested and connected.

##### Week 38th **30 September - 6 October.**

## Summary

As deadlines approach, we looked into the option of synthesizing new primers to
fix a DNA sequence problem in our Biobricks. The Wiki Freeze being so close we started uploading all our results to our wiki. We had a lot of stress going on and ran the final tests on our project. We were both exhausted and excited to reach the conclusion of this great experience as a team.

### Wet lab

Nanoparticles were observed in Nanosight with the encapsulation of BSA to
confirm the size. Some liberation assays were done to quantify protein
concentration with PBS at pH 7.4 to simulate physiological medium.
For the parts, a PCR was done.
Some gels were run.
A sequencing company was contacted to troubleshoot our sequence.

### Software

The software design was defined and most of the databases completed.

* ![IDT logo](https://static.igem.org/mediawiki/2018/7/72/T--TecCEM--Sponsors2.png)
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# Team:TecCEM/Experiments

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  [Parts](https://2018.igem.org/Team%3ATecCEM/Parts)
  [Basic Part](https://2018.igem.org/Team%3ATecCEM/Basic_Part)
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  Part](https://2018.igem.org/Team%3ATecCEM/Composite_Part)
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  [Human Practices](https://2018.igem.org/Team%3ATecCEM/Human_Practices)
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  Engagement](https://2018.igem.org/Team%3ATecCEM/Public_Engagement)
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  [Team Members](https://2018.igem.org/Team%3ATecCEM/Team)
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![Cell Gif](https://static.igem.org/mediawiki/2018/4/41/T--TecCEM--Cells--Experiments.gif)

# Experiments

### Project/ Experiments

[Index](#experimentsSubmenu)

* Description
* [Protein](#proteinSubmenu)
  + [Chitosan](#chitosan)
    - Protein encapsulation protocol
    - Protein encapsulation efficiency
      protocol
    - Protein liberation and stability
      protocol
  + [Polyacrilamide gel](#polyacrimlamide-gel)
    - AB Polyacrilamide gel
    - Standard polyacrylamide gel
  + [Production/purification](#productpurification)
    - Criofracture
    - Fluorescent protein production
    - Protein production/purification
      protocol
  + [Proteic quantification](#proteicquantification)
    - Bradford
    - Lowry
    - Pierce BCA
* [Cell](#cellSubmenu)
  + In vitro burn essay protocol
* [Reactants](#reactantsSubmenu)
  + Protocol for the creation of lysis buffer P
  + Protocol for the creation of polyacrylamide gel
  + Protocol for the creation of thermal lysis buffer
  + Protocol to make polyacrylamide AB
* [Nucleic Acids](#nucleicSubmenu)
  + [Cell Transformation](#celltransformation)
    - Chemocompetent cells transformation protocol
    - Protocol for chemocompetent cells
    - Thermal Shock cell transformation protocol
  + [Enzyme restriction](#enzymerestriction)
    - Protocol for enzyme digestion/alkaline phosphatase treatment
      of iGEM plasmids and ligation
    - Protocol for enzyme ligation of iGEM parts
    - Protocol for enzyme restriction of IDT parts and iGEM plasmid
  + [PCR](#pcr)
    - Protocol for Colony PCR
    - Protocol for PCR using VR and VF2 primers
  + [Plasmid extraction](#plasmidextraction)
    - Agarose Band purification (GenElute - Sigma)
    - Alkaline plasmid purification protocol
    - MiniPrep Sigma extraction protocol

This is our experiment section. Here we compile important protocols for the development of
TecTissue, ranging from our bacterial transformation procedures to our cell proliferation
assays.
We also address cell culture maintenance and protein loaded chitosan nanoparticles.
Here you may find the protocol for our growth factor delivery to damaged cells and how much
harm can be inflicted in vitro.

# Protein

## Chitosan nanoparticles

[### Protein encapsulation protocol](#encapsulation)

[### Protein encapsulation efficiency protocol](#encapsulation-efficiency)

[### Protein liberation and stability protocol](#liberation-and-stability)

## Polyacrylamide gel

[### AB Polyacrilamide gel](#abpolyacrilamidegel)

[### Standard polyacrylamide gel](#stdpolyacrilamidegeel)

## Production/purification

[### Criofracture](#criofracture)

[### Fluorescent protein production](#fluorescentproteinproduction)

[### Protein production/purification protocol](#proteinproductionpurificationprotocol)

## Proteic quantification

[### Bradford](#bradford)

[### Lowry](#lowry)

[### Pierce BCA](#piercebca)

# Cells

[### In vitro burn essay protocol](#cellessay)

# Reactants

[### Protocol for the creation of lysis buffer P](#creationlysisbbuffer)

[### Protocol for the creation of polyacrylamide gel](#creationpolyacrylamidegel)

[### Protocol for the creation of thermal lysis buffer](#creationthemlysisbuffer)

[### Protocol to make polyacrylamide AB](#creationpolyacrylamideab)

# Nucleic acids

## Cell transformation

[### Chemocompetent cells transformation protocol](#chemocompetentcells)

[### Protocol for chemocompetent cells](#pcc)

[### Thermal Shock cell transformation protocol](#tsc)

## Enzyme restriction

[### Protocol for enzyme digestion/alkaline phosphatase treatment of iGEM plasmids and ligation](#ligacion)

[### Protocol for enzyme ligation of iGEM parts](#enzymelig)

[### Protocol for enzyme restriction of IDT parts and iGEM plasmid](#enzres)

## PCR

[### Protocol for Colony PCR](#protocolforcolonypcr)

[### Protocol for PCR using VR and VF2 primers](#protocolforpcrvrvf2primers)

## Plasmid extraction

[### Agarose Band purification (GenElute - Sigma)](#agarosebandpurification)

[### Alkaline plasmid purification protocol](#alkalineplasmidpurification)

[### MiniPrep Sigma extraction protocol](#miniprepsigma)

* ![IDT logo](https://static.igem.org/mediawiki/2018/7/72/T--TecCEM--Sponsors2.png)
* ![Matlab logo](https://static.igem.org/mediawiki/2018/d/d1/T--TecCEM--Sponsors5.png)
* ![Tec Monterrey logo](https://static.igem.org/mediawiki/2018/b/b4/T--TecCEM--Sponsors4.png)
* ![CBC Cells logo](https://static.igem.org/mediawiki/2018/4/4a/T--TecCEM--Sponsors1.jpeg)
* ![NEB logo](https://static.igem.org/mediawiki/2018/a/aa/T--IISER-Bhopal-India--sponsorNeb.png)
* ![Snapgene logo](https://static.igem.org/mediawiki/2018/f/f6/T--Cornell--SnapGeneLogo.jpg)
* ![Uniparts logo](https://static.igem.org/mediawiki/2018/4/4f/T--TecCEM--Sponsors6.jpeg)
* ![LEI logo](https://static.igem.org/mediawiki/2018/2/2d/T--TecCEM--Sponsors3.png)
* ![MBM logo](https://static.igem.org/mediawiki/2018/2/2f/T--TecCEM--Sponsors8.png)
* ![VM Sol logo](https://static.igem.org/mediawiki/2018/c/cd/T--TecCEM--Sponsors7.jpeg)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:TecCEM/Results

[![Tec Tissue Logo](https://static.igem.org/mediawiki/2018/3/3f/T--TecCEM--TecTissue.png)](https://2018.igem.org/Team%3ATecCEM)
[![Tec Tissue Logo](https://static.igem.org/mediawiki/2018/5/54/T--TecCEM--TeamLogo.png)](https://2018.igem.org/Team%3ATecCEM)

* [Home](https://2018.igem.org/Team%3ATecCEM)
* Project

  [Description](https://2018.igem.org/Team%3ATecCEM/Description)
  [Design](https://2018.igem.org/Team%3ATecCEM/Design)
  [Results](https://2018.igem.org/Team%3ATecCEM/Results)
  [Experiments](https://2018.igem.org/Team%3ATecCEM/Experiments)
  [Hardware](https://2018.igem.org/Team%3ATecCEM/Hardware)
  [Software](https://2018.igem.org/Team%3ATecCEM/Software)
  [InterLab](https://2018.igem.org/Team%3ATecCEM/InterLab)
  [Model](https://2018.igem.org/Team%3ATecCEM/Model)
  [Demonstrate](https://2018.igem.org/Team%3ATecCEM/Demonstrate)
  [Improve](https://2018.igem.org/Team%3ATecCEM/Improve)
* Parts

  [Parts](https://2018.igem.org/Team%3ATecCEM/Parts)
  [Basic Part](https://2018.igem.org/Team%3ATecCEM/Basic_Part)
  [Composite
  Part](https://2018.igem.org/Team%3ATecCEM/Composite_Part)
* Human Practices

  [Human Practices](https://2018.igem.org/Team%3ATecCEM/Human_Practices)
  [Public
  Engagement](https://2018.igem.org/Team%3ATecCEM/Public_Engagement)
* [Notebook](https://2018.igem.org/Team%3ATecCEM/Notebook)
* Team

  [Team Members](https://2018.igem.org/Team%3ATecCEM/Team)
  [Collaborations](https://2018.igem.org/Team%3ATecCEM/Collaborations)
  [Attributions](https://2018.igem.org/Team%3ATecCEM/Attributions)
* [Safety](https://2018.igem.org/Team%3ATecCEM/Safety)
* Awards

  [Hardware](https://2018.igem.org/Team%3ATecCEM/Hardware)
  [Software](https://2018.igem.org/Team%3ATecCEM/Software)
  [Measurement](https://2018.igem.org/Team%3ATecCEM/Measurement)
  [Model](https://2018.igem.org/Team%3ATecCEM/Model)

![Cell Gif](https://static.igem.org/mediawiki/2018/a/aa/T--TecCEM--Cells.gif)

# Results

### Project/ Results

[Index](#experimentsSubmenu)

* Overview
* Summary
* First phase
* Second phase
* Third phase
* Burn assay
* Burn assay with treatment
* Cell culture in BOB

## Overview

To accomplish our project objective several recombinant proteins must be produced in order to
analyze
their effects on a co culture of fibroblasts (L929) and mesenchymal cells after an in vitro
burn assay.
To evaluate the efficiency of the treatment, a measurement of the proliferation rate was
performed
using LDH (lactate dehydrogenase) as an analysis metabolite. Given that our treatment involves
the
usage of a growth factor, the rate at which this protein is released into the medium is
critical to
avoid adverse effects in the cellular line (such as cancer), for that reason a nano
encapsulation with
chitosan was also performed to control the rate at which our growth factor is released.

## Summary

### What did we accomplish?

* Nanoencapsulation in chitosan of leptin, BSA and RFP.
* Realization of in vitro burn assay.
* Realization of leptin proliferation essay.
* Obtaining parts with adequate enzyme recognition sites.
* Protein production of leptin and tenascin C (More validations are needed).
* Co-culture of fibroblast and mesenchymal cells.
* Cellular growth in TaCO-BOB hardware.

### What happened?

* We started ligating parts, no bands of expected size were observed but we attributed this
  fact to low sensitivity of agarose gels, so we proceeded with transformation and protein
  induction. Nevertheless, we realized that consistent results were not achieved (bad protein
  migration rates, sometimes no induction band could be observed). [Unfortunately, we
  invested a lot of time on this period]
* Then we started thinking that our parts did not had the necessary extra nucleotides for
  proper cleavage of restriction enzymes and what we were truly observing was the effect of
  star activity and inappropriate ligation, resulting in non specific plasmids which did not
  contained our whole part but conferred the bacteria antibiotic resistance.

### How did we solved it?

We designed primers that incorporate the necessary nucleotides into our parts. Thus, we started
having proper migrations of project parts and posterious transformations and protein production
of BBLEP.

### How should we improve it?

By including better protein reporters for the screening of transformed, functional bacteria
colonies.

Our proyect is divided into three sections which are the following:

1. Phase 1: *E. coli* transformation
2. Phase 2: Protein production
3. Phase 3: Test in cellular line

# Phase 1 *E. coli transformation*

Once our IDT synthesis arrived, we set the objective of ligating the composite parts of
tenascin ([BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)) [also called
BBTNC], leptin ([BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)) [also
called BBLEP] and
collagen ([BBa\_K2719007](http://parts.igem.org/Part%3ABBa_K2719007)) [also
called BBCOL], into an RFC10 compatible psB1C3 iGEM plasmid
for future transformations into *E. coli*. We had several transformation attempts
(Figure 1),
most of the times there are no adequate migrations patterns achieved. We
carried on with
bacterial transformation and protein production, as we thought that bands of
ligation could
not be seen given the agarose sensibility, surprisingly bacterial colonies
(either *E. coli*
BL21(DE3) and DH5 alpha ) were seen (Figure 2) in selective medium but no
constant protein
production could be achieved (Go to phase 2for more information). After
many failed
attempts and a lot of time invested, we suspected that maybe ligations were
failing due to
the absence of “close to the end DNA cleavage nucleotides” (needed for
restriction
cutting). To solve the problem previously mentioned, primers were designed to
tackle this
problems using iGEM prefix and suffix sequences as template for amplification.
We amplified
BBLEP (Figure 3) and ligated it into psB1C3 finally obtaining the expected
sizes at each
step. Thus with this methodology we could resolve the difficulties for BBLEP
and achieving
a transformation in BL21(DE3) (Figure 4), due to timewise limitations and the
embedded DNA
synthesize problems, BBCOL and BBTNC could not be tested with this methodology.
Future
plans include applying that methodology to those parts and transforming into
respective
bacteria strains.

![Clave 1](https://static.igem.org/mediawiki/2018/4/48/T--TecCEM--Results01.jpg)

Figure 1. First attempt of ligation of IDT parts
into a
psB1C3 plasmid

* **Experiment:** Electrophoresis.
* **Matrix:** Agarose (0.85%).
* **Conditions:** 100 volts, 60 minutes in TBE buffer.
* The samples were charged in the following order:
  1. 2-log DNA ladder (6uL)
  2. BBLEP 2739bp (3uL + 3uL of loading buffer)
  3. BBCOL 3566bp (3 uL + 3uL of loading buffer)
  4. BBTNC 3196bp (3 uL + 3uL of loading buffer)
  5. BBTNC 3196bp (3 uL + 3uL of loading buffer)
  6. 2-log DNA ladder (6uL)
  7. 1kb DNA ladder (6uL)

![Clave 2](https://static.igem.org/mediawiki/2018/2/20/T--TecCEM--Results02.jpg)

Figure 2. Culture plates containing transformed *E.
coli*
.1) *E. coli* DH5 alpha containing BBCOL 2) *E. coli* DH5 alpha containing
BBLEP 3) *E.
coli*
DH5 alpha containing BBTCD.

![Clave 3](https://static.igem.org/mediawiki/2018/1/1c/T--TecCEM--Results03.jpg)

![Clave 3](https://static.igem.org/mediawiki/2018/0/06/T--TecCEM--ResultsClave3.png)

Figure 3. BBLEP amplicon obtained after PCR with
designed primers

* **Experiment:** Electrophoresis
* **Matrix:** Agarose (0.85%)
* **Conditions:** 100 volts, 60 minutes in TBE buffer
* The samples were charged in the following order:
  1. 2-log DNA ladder (6uL)
  2. BBLEP PCR amplicon (3 uL + 3uL of loading buffer)

![Clave 4](https://static.igem.org/mediawiki/2018/7/75/T--TecCEM--Results04.jpg)

Figure 4. Culture plates containing transformed *E.
coli* BL21(DE3) with BBLEP.

# Phase 2 *Protein production*

After doing transformation into *E. coli* BL21 (DE3) we started the production and
purification of our protein of interests. This phase was a key factor for the demonstration
of the functionality of our project. First, we obtained polyacrylamide gels that had clear
signs of maximum induction of our proteins at 5 hours after IPTG induction. The results
that we obtained were inconsistent between several repetitions of the induction.

After that observation several hypotheses were formulated in order to explain those events,
the first one was that the synthesis sequence had several errors, which made the peptide
product unstable, and an easy target for degradation. This hypothesis was mainly supported
by the apparition of the protein at 5h after induction, but its complete disappearance
after 16 hours, specifically TCD that needs a protein fusion to improve its stability and
reduce the rate of degradation.

The second hypothesis was that the used strain (*E. coli* BL21 (DE3)) was mislabeled,
therefore the transformation and induction were on strains unable to produce RNApol T7.
This idea was discarded after several experiments were made on fresh strains, obtaining the
same inconsistent results. Even though the results continued to be inconsistent, with the
fresh strains some improvement was made. Tenascin was observed for the first time on
SDS-PAGE after 5h of induction (see figure 5). Also a Dot Blot was performed in order to
evaluate the presence of Tenascin and Leptin using the His-TAG (see figure 7).

The third and last hypothesis was that an error on the synthesis meant that there were not
enough nucleotides for allowing the restriction enzymes (EcoRI and PstI) to work properly,
reducing ligation efficiency. In order to test this hypothesis new primers were developed,
those primers after amplification with PCR, added extra nucleotides to the prefix and
suffix o the parts promoting the properly work of the enzymes.

Finally, since *E.coli* BL21 (DE3) has a protein that migrates at the same levels [1]
as those
expected for leptin, a Western Blot was performed in order to get more specific results.
Using an anti-His antibodies we were able to determined the presence and the production of
the leptin. In order to do this, a inoculum of transformed*E.coli* BL21 was induced at
a
concentration of 1mM IPTG. The samples were lizated utilizing RIPA buffer and were loaded
on an gradient 5%-20% BioRad SDS-PAGE. This was transferred to a nitrocellulose membrane
and incubated with anti-His antibodies. The results were positive, obtaining bands (where
the antibody binds) at a size close to 17 kDa, similar to the expected leptin molecular
weight (see figure 8). So the production of leptin is proposed, but further experimentation
is required to standardize its production.

## Tenascin confirmation (protein level)

![Clave A](https://static.igem.org/mediawiki/2018/8/8d/T--TecCEM--ResultsClaveA.png)

Figure 5. Analysis of BBLEP and BBTCD before IPTG
induction

* **Experiment:** SDS-PAGE
* **Matrix:** Polyacrylamide (15%)
* **Conditions:** 1080 volts for 20 minutes, 120 volts 60 minutes.
* **Objective:** Determine the presence of protein from BBLEP (BBa\_K2719009)
  and
  BBTCD (BBa\_K2719005) in protein producer strain *E. coli* BL21 (DE3)
* **Description:** Crude lysate (30 uL + 10 uL laemmli buffer) coming from
  transformed BL21 (DE3) strains and induced with 1 mM IPTG for different hours at 37
  Celsius
  degrees were loaded onto polyacrylamide gel and runned with SDS-Tris-Gly buffer
* The samples were charged in the following order:
  1. BBTCD V2 (At 16 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  2. BBTCD V1 (At 16 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  3. BBLEP V2 (At 16 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  4. BBLEP V3 (At 16 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  5. BBTCD V1 (At 16 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  6. BBLEP V1 (At 16 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  7. BBTCD V2 (At 5 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  8. BBTCD V1 (At 5 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  9. BBLEP V2 (At 5 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  10. Protein Ladder
* **Analysis:** The gel has a constant band pattern among lanes, which
  represents the native proteins of BL21 (DE3) strain. On the other hand, a single band
  is highly ovestanding which can be seen in lane 7, here a single pattern band between
  25 and 37 kDa correspond to a possible apparition of BBTCD protein, which has a
  theoretical weight of 35.1 kDa, the band stand as very likely candidate as its form and
  luminosity correspond to a common protein band derived from an IPTG expression,
  nevertheless, at 15 hours the same part (lane 1) shows no sign of the protein, this can
  be explained as the lost of function of IPTG given by continuous exposure to high
  temperatures and consequently loss of protein production possibility and gradual
  degradation of existing protein. For BBLEP no sign of protein production could be
  recorded. More analysis to BBTCD must be done to corroborate the existence of protein,
  and a restructure of troubleshooting for BBLEP must be done to create better results of
  production..

## Selection of Tenascin producer strain

* **Experiment:** SDS-PAGE
* **Matrix:** Polyacrylamide (20%)
* **Conditions:** 80 volts for 20 minutes, 120 volts 60 minutes.
* **Objective:** Determine the *E. coli* BL21 (DE3) strain that produce
  the
  protein BBTCD (BBa\_K2719005).
* **Description:** Crude lysate (30 uL + 10 uL laemmli buffer) coming from
  transformed BL21 (DE3) strains and induced with 1 mM IPTG for different hours at 37
  Celsius degrees were loaded onto polyacrylamide gel and runned with SDS-Tris-Gly
  buffer.
* The samples were charged in the following order:
  1. BBTCD V1 (At 5 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  2. BBTCD V3 (At 5 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  3. BBTCD V2 (At 5 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  4. Negative control *E.coli* BL21 (DE3) without transformation (At 5 hours of
     induction)
  5. Protein Ladder
* **Analysis:**Analysis: A single band is highly overstanding which can be
  seen in lane 3, here a single band of 25 kDa that confirm the possible presence of
  BBTCD protein in the BBTCD V2 strain, furthermore there is an inconsistency in this
  line because the theoretical weight of TCD is 35.1 kDa. Obtaining an unexpected
  molecular weight for BBTCD as a fusion protein, made us suggested that the protein
  sequence is incomplete. This aceveración can explain the reason why there is no
  presence of BBTCD at 16 hours of induction. The GST as a fusion protein helps for the
  stability of the entire protein. Its absence difficult to have the protein in the
  stable form and it is possibly degraded.

![Clave B](https://static.igem.org/mediawiki/2018/e/ed/T--TecCEM--ResultsClaveB.png)

Figure 6. Analysis of BBTCD in different *E. coli*
BL21 strains

## Tenascin and leptin confirmation (protein level)

![Clave C](https://static.igem.org/mediawiki/2018/0/04/T--TecCEM--ResultsClaveC.png)

Figure 7. Dot Blot analysis of BBTCD and BBLEP

* **Experiment:** Dot blot
* **Matrix:** Nitrocellulose membrane
* **Conditions:** Incubation with Anti His tag antibody (dilution 1:1000)
  for 1 hour and revelation by Dye Green transilluminator protocol.
* **Objective:** Determine the presence of protein from BBLEP (BBa\_K2719009)
  and BBTCD (BBa\_K2719005) in protein producer strain *E. coli* BL21 (DE3)
* **Description:** Crude lysate coming from transformed BL21 (DE3) strains
  and induced with 1 mM IPTG for 5 hours at 37 Celsius degrees were loaded onto
  nitrocellulose membrane (3 uL) and incubated with Anti His tag antibody coupled with a
  fluorochrome reporter.
* The samples were charged in the following order:
  1. Negative control *E.coli* BL21 (DE3) without transformation (At 3 hours of
     induction)
  2. Negative control *E. coli* BL21 (DE3) without transformation (At 5 hours
     of
     induction)
  3. BBTCD V2(At 3 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  4. BBTCD V2(At 5 hours of induction) [[BBa\_K2719005](http://parts.igem.org/Part%3ABBa_K2719005)]
  5. BBLEP V1 (At 3 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  6. BBLEP V1 (At 5 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  7. BBLEP V2 (At 3 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  8. BBLEP V2 (At 5 hours of induction) [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)]
  9. Positive control
  10. Nothing
* **Analysis:** After the inconsistent results obtained in the
  polyacrylamide gels, a Dot Blot was executed. Observation of colored points in
  positions 3, 5 an 7 gives a positive result in presence of recombinant proteins which
  contain a histidine tag, in positions 4, 6 and 8 an even more colored point is
  detected, this confirm that the production of protein increases significantly after 5
  hours of induction. On the other hand positions 1 and 2, which accounts for negative
  control, resolves that *E. coli* BL21 (DE3) does not produces endogenous native
  proteins
  with a high content of histidine tags that could be misunderstood as our protein, this
  event partially eliminates false positive results. Meanwhile position 9, which account
  for positive control, shows no coloured point, this effect must be due to the high
  degree of dilution that we applied to the positive control (1:1000), also there is no
  visualization of the mark that normally happens when the sample dries, thus we can
  conclude that our positive control, which was a recombinant protein from E coli, was
  not in optimal state. This result complicates the final decision about our protein
  production, for that more dot blots or even western blots must be applied for a better
  confirmation of results.

![Clave C](https://static.igem.org/mediawiki/2018/0/01/T--TecCEM--ResultsClaveW.png)

Figure 8. Western Blot analysis of newly ligated
BBLEP [[BBa\_K2719009](http://parts.igem.org/Part%3ABBa_K2719009)].

* **Experiment:** Western blot
* **Matrix:** Nitrocellulose membrane
* **Conditions:** Incubation with Anti His tag antibody (dilution 1:1000)
  for overnight.
* **Objective:** Determine the presence of protein from newly ligated BBLEP
  (BBa\_K2719009) in protein producer strain *E. coli*BL21 (DE3).
* **Description:** CCrude lysate coming from transformed BL21 (DE3) strains
  and induced with 0.5 mM IPTG for 5 hours at 37 Celsius degrees were loaded onto
  nitrocellulose membrane (3 uL) and incubated with Anti His tag antibody coupled with a
  fluorochrome reporter. At left SDS-PAGE 15% gel revealed with Coomassie blue for 30
  minutes, at right western blot.
* The samples were charged in the following order:
  1. Molecular weight ladder Spectra.
  2. BBLEP (At 0 hours of induction)
  3. BBLEP (At 2 hours of induction)
  4. BBLEP (At 4 hours of induction)
  5. BBLEP (At 6 hours of induction)
* **Analysis:**As is observed on the SDS-PAGE, is almost impossible to
  distinguish between the native E.coli BL21 (DE3) protein and leptin, as both proteins
  migrate in a similar way, therefore both bands render undistinguishable. To overcome
  this problem, a Western Blot was performed using an anti-His antibody, taking advantage
  that leptin contains a 6xHis Tag and the native protein does not, so the antibody will
  only bind to leptin. The results after the incubation with the antibodies determined
  not only the presence of a peptide of around 17 kDa with an histidine Tag, presumably
  leptin. But also as the width of the band increased as the induction time increased
  (for times 0, 2h, 4h and 6h), thus suggesting a successful IPTG induction of the part.

As is shown on the Western Blot (see figure 8) recombinant leptin was successfully produced
by our parts in *E.coli* BL21 (DE3), but the degree of purity obtained was not
sufficient for
its usage on a cell line. But, as a proof of concept was necessary to demonstrate the
effect of recombinant leptin on cell proliferation in burn damaged tissue. Then, we decided
to use high degree of purity commercially available leptin for our assays on cell lines. In
this way, the functionabillity of our project is demonstrated on a real experimental
manner, minimizing the effect of external variables (such as contamination from undesired
components).

# Phase 3: *Nanoencapsulation and test in cellular line*

As a delivery vehicle was needed protein-loaded chitosan nanoparticles were prepared by
ionic gelation method. For a correct standardization of the encapsulation protocol, we used
BSA as protein. BSA is often employed for concentration measurements in protein assays,
including encapsulation efficiency and protein release in a solution of loaded
nanoparticles.

Chitosan nanoparticles were prepared with a pH of 5.0 since BSA has an isoelectric point of
4.7 at 25 °C with TPP polyanion under mild conditions following our standardized protocol.

## What we expected:

We wanted to create an efficient drug delivery method that would regulate leptin liberation
into the cells as an uncontrolled amount of leptin can cause a swelling reaction. By
standardizing the nanoparticle particle size, particle count, encapsulation efficiency, and
protein release behavior in a physiological pH solution we expected to accurately predict a
cell proliferation increase in treated cell lines with nanoparticles against non-treated.

## What we achieved: *Characterization of nanoparticles*

For the encapsulation of BSA at pH at 5.0 a particle analysis in NanoSight NS300 to obtain
the particle size distribution was performed. A dot graph for a triplicate analysis is
presented below.

### Particle size and particle count

Particles were analyzed in NanoSight NS300, from which we obtained 127.8 nm as mean particle
size with a broad standard deviation of 90.1 nm. NanoSight NS300 displays a video when
analyzing a sample volume of nanoparticle solution, which we include here.

![Clave 4](https://static.igem.org/mediawiki/2018/f/f9/T--TecCEM--Results31.png)

Figure 9. RFP loaded nanoparticles graphs of
concentration (particles/mL) vs Size (nm) obtained from NanoSight NS300

[

](https://static.igem.org/mediawiki/2018/8/88/T--TecCEM--Chitosan.mp4)

Figure 9. RFP loaded nanoparticles graphs of
concentration (particles/mL) vs Size (nm) obtained from NanoSight NS300

## Analysis:

Some chitosan nanoparticles displayed a bigger diameter, this might be a result from the
swelling of chitosan layer and aggregation of single particles while dispersed in water,
causing a detection of several bigger particles, shifting the mean value and standard
deviation. Transmission electron microscopy (TEM) was used as a second method of
confirmation, in which nanoparticle morphology was observed. We think this difference is
caused by the sample treatment required for TEM, that enables adequate particle
distribution to observe them individually. A mean size by TEM was determined to be 27.7 nm.

### Encapsulation efficiency

A Bradford assay was performed to determine the protein encapsulation percentage in order to
evaluate the efficiency of the protocol used to form loaded chitosan nanoparticles. The
recovery of protein from the encapsulation procedure was 42% at the stated conditions.

### Protein release behavior

We established the relation between the amount of protein released by nanoparticles with
respect to time, measuring the protein concentration at 0, 2, 4, 12, 18 and 24 hrs.
Nanoparticles were incubated at 37 °C and 100 rpm suspended in PBS pH 7.4 to estimate how
they would behave in a physiological medium. After 24 hours almost 50% of the protein was
released.

![Clave 4](https://static.igem.org/mediawiki/2018/f/f3/T--TecCEM--Results32.png)

Figure 10. Protein release percentage according to the
initial amount of encapsulated protein. The assay was performed with individual
aliquots for every point in time.

### Leptin loaded chitosan nanoparticles action in cell lines

As the main target of this project was to prove protein release from a chitosan
encapsulation that would increase cell proliferation in a cell line, we encapsulated leptin
under the same conditions. Afterward, leptin-chitosan nanoparticles were tested in L929
fibroblasts cell line which was damaged with a hot solution at different degrees. For every
degree of damage, a well was supplied with 3.4 ng/mL of leptin growth factor. Results
analyzed after 72 h are presented below. Commercial leptin growth factor effect on burned
L929 fibroblasts is demonstrated through the performance of this experiment.

# Cell line burn assay standardization

Burn injuries represent a serious issue that involves pathophysiological changes, it is
important to develop new mechanisms to test the viability of improvements or new
treatments. For that reason,we decided to perform an assay which consisted of analysing
cell proliferation after submitting L-929 cell line to a burning procedure. This allowed us
to monitor if the evaluation of our treatment through this mechanism was feasible.

After setting different exposure times (15, 30, 45 and 60 seconds) to which each well would
be exposed to HBSS (Hank solution) at 60ºC, we registered the results comparing the damage
taken by the cell culture in each of the wells. We can conclude that the longer the cells
are exposed to the solution, the more damage they take and the less cellular density is
observed. This analysis allowed us to proceed with the evaluation of TecTissue performing
another burn assay to apply later the treatment we designed. The importance of the
standardization lies in the fact that it establishes the way in which we will perform the
following assays to determine the efficiency of our project and set the basis for a more
specific complex burn model.

In this in vitro burn model, it is possible to observe the increasing cell injury and
significant changes in cell viability and morphology in relation to the time they were
exposed to the hot HBSS.

![Clave 4](https://static.igem.org/mediawiki/2018/2/2b/T--TecCEM--Demonstrate1.png)

Figure 11. Decrease in cellular density according to the
exposure time to hot HBSS.
NB - No burn

In conclusion, the exposure of the culture to hot HBSS caused visible damage to the cells,
causing possible cell necrosis and cell detachment of cell culture flasks. The different
times employed are directly proportional to the harm taken by the culture and therefore the
number of cells is visibly reduced. This assay allows researchers to perform in vitro
analysis of burn process and the nature of the procedure makes it a feasible and
reproductive alternative to study the cellular and molecular alterations throughout the
burn process. In perspective, we could measure necrosis, cell viability, LDH, among others.

# Burn assay with treatment

Once the burn assay was standardized, it was time to prove the efficiency of TecTissue. For
that reason, we performed another burn assay with the times stated before but applying the
hot HBSS to a co-culture of L-929 fibroblasts and human mesenchymal stem cells. Since our
project is based on the combination of several factors, we decided to test the activity of
each throughout the regeneration of the cultures.The compatibility of both cell lines
allowed us to perform the procedure without any complication. However, due to the
importance of the procedure, an MTT assay was performed additionally to ensure the accuracy
of the results obtained and to legitimate the conclusions reached after the assay. The
following pictures show the amount of cells depending on three factors:

* Time of exposure to hot HBSS
* Treatment given after burning the co-culture
* + Chitosan nanoparticles with leptin encapsulated
  + Empty chitosan nanoparticles
* Co-culture of fibroblasts and mesenchymal cells

According to the figures shown, the damage taken by the cells is visible related to the
amount of time they were exposed to the hot solution; however, in order to determine at a
molecular level the viability of the cells remaining, we performed an MTT assay with the
purpose of relating the proliferation of the cells with the efficiency of the treatment.

![Clave 4](https://static.igem.org/mediawiki/2018/6/6d/T--TecCEM--RC34.png )

Figure 12. Proliferation Assay in different hot HBSS
exposure. A and B; 48hrs and 72hrs post burn assay, with Chitosan charged with leptin
and empty nanopaticles (NP). C; comparison of proliferation with leptin Chitosan NP.

This figure compares the proliferation of the co-culture when one of the samples is treated
with all the elements that form part of TecTissue (collagen, heparan, leptin nanoparticles)
and the other with empty nanoparticles. The graph shows that the dosified release of leptin
increases the proliferation and regeneration of the cells while the ones that received the
treatment without the leptin did not have the same response. This behaviour is due to the
activity of the leptin, which is slowly released into the matrix allowing cells to absorb
it and enhance their metabolic activity to proliferate. Nevertheless, we can observe that
empty chitosan nanoparticles may cause some response.

![Clave 4](https://static.igem.org/mediawiki/2018/8/83/T--TecCEM--Demonstrate2.png)

Figure 13. Increase in cellular density based on the
application of chitosan nanoparticles charged with leptin. Compa rison of different
exposure times 48 hours post burn assay.

After analysing the results obtained from the MTT assay, we confirmed our hypothesis and
observed that cells treated with TecTissue were indeed metabolic enhanced and confluent
compared to the ones that were not treated. Some aspects to consider are that even though
the leptin, heparan, and collagen were studied, none of them were evaluated separately,
which means that further analysis should be performed to determine the activity of each
element within the treatment. As stated in the corresponding sections, chitosan, heparan
and collagen each play a specific role in our project and the combination of them represent
at first glance, a good opportunity to develop new treatments for burn injuries. Even
though we demonstrated the efficiency of TecTissue, it is important to mention that within
72 hours, the confluence of both conditions (charged and empty NP), present no significant
differences. This means that the effects of our treatment are more visible within the first
48 hours. Since confluence of both wells were similar, we can say that TecTissue is a
feasible treatment but within the short term. Further analysis should be made to determine
whether it is possible to increase the efficiency of the treatment considering the areas of
opportunity identified after the assay.

Another thing that needs to be sorted out, is the relation between the co-culture and the
damage induced, with the presence or absence of leptin within the nanoparticles because the
results obtained with the microscope suggest that the chitosan alone could plays a key role
in the regeneration and proliferation of the fibroblasts.

![Clave 4](https://static.igem.org/mediawiki/2018/c/ca/T--TecCEM--RC36.png)

Figure 14. Similar confluence in both conditions 72
hours post burn assay.

Furthermore, it is necessary to determine the efficiency of the project, measuring
metabolites such as Lactate Dehydrogenase (LDH), proliferation cytokines like tissue growth
factor beta, which is known to be released by the mesenchymal stem cells due to the action
of leptin.

The amount of cells that can be seen in each well of the plate are directly related to the
time it was exposed to the hot HBSS, the treatment employed and the time it was left
incubated afterwards. For that reason, it is important to describe the role of each element
of the treatment and its influence in the regeneration of the cells: Collagen and heparan
as the scaffold, leptin as the growth factor and chitosan as the vehicle that regulated the
release of the drug. The combination of all these, allowed cells to react positively and
enhance the proliferation, increasing its rate and potentiating the healing process.

# Cell culture in BOB

Part of the evaluation of TecTissue consisted in testing the feasibility of our hardware,
which was designed with the purpose of automatizing 2D/3D cell cultures. In order to
evaluate if BOB is useful, we decided to perform a co-culture within it and analyse if
cells were able to adhere to the PDMS surface and proliferate. The following pictures
display how cells behaved when cultivated in BOB. To achieve our goal, we had to adequate
the hardware with the molecules necessary for the correct adherence of the cells. In other
words, we tested several combinations of the elements that comprise TecTissue. For example,
a BOB with a scaffold made out of PDMS supplemented with collagen and heparan. The same
study was performed with other combinations of said elements and removing the PDMS. After
incubating for at least one day, we photo documented and recorded the results observed in a
microscope employing fluorescence to ensure cells were correctly adhered to the synthetic
matrix.

The results demonstrate that porous-collagen-based scaffolds membrane are effective for cell
culture and tissue engineering. The images obtained from the epifluorescence microscope
with a mitochondrial dye allow us to conclude that employing these elements it is possible
to perform a co-culture. This is supported by the immunofluorescence assay that allowed us
to see the correct attachment of the cells. Further analysis should be performed to enhance
the efficiency of the PDMS and increase the cellular adhesion.

![Clave 4](https://static.igem.org/mediawiki/2018/b/b8/T--TecCEM--Demonstrate3.png)

Figure 15. Cell cultures in the hardware. BOB 1 contains
heparan sulfate, collagen, co-cultured cells and PDMS, BOB 2 (heparan sulfate and
co-cultured cells), BOB 3 (collagen and co.cultured cells) and BOB 4 (heparan sulfate,
collagen and co-cultured cells).

* ![IDT logo](https://static.igem.org/mediawiki/2018/7/72/T--TecCEM--Sponsors2.png)
* ![Matlab logo](https://static.igem.org/mediawiki/2018/d/d1/T--TecCEM--Sponsors5.png)
* ![Tec Monterrey logo](https://static.igem.org/mediawiki/2018/b/b4/T--TecCEM--Sponsors4.png)
* ![CBC Cells logo](https://static.igem.org/mediawiki/2018/4/4a/T--TecCEM--Sponsors1.jpeg)
* ![NEB logo](https://static.igem.org/mediawiki/2018/a/aa/T--IISER-Bhopal-India--sponsorNeb.png)
* ![Snapgene logo](https://static.igem.org/mediawiki/2018/f/f6/T--Cornell--SnapGeneLogo.jpg)
* ![Uniparts logo](https://static.igem.org/mediawiki/2018/4/4f/T--TecCEM--Sponsors6.jpeg)
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* ![MBM logo](https://static.igem.org/mediawiki/2018/2/2f/T--TecCEM--Sponsors8.png)
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