iGEM 2017 — Team UPMC_PARIS

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# Team:UPMC PARIS/Notebook

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* [Judging form](https://igem.org/2017_Judging_Form?team=UPMC_PARIS)

# Notebook

### Protocols iGEM Team 2017 UPMC-Paris

#### Bacterial competentiation

We started from frozen E. coli DH5α which we thaw on ice and then streak on Lysogeny Broth
(LB) agar plates (autoclaved 20 g sterile agar for 1L distilled water) that we incubated
overnight at 37°C. The next day we inoculated one colony on 4 mL LB (autoclaved 25g LB in
distilled water), overnight at 37°C and 220 rpm. The following day, we inoculated 500µL of
these pre-culture in 50 mL of LB and incubated them at 37°C until OD600: 0,5. At this stage,
bacterial cells are centrifuged (10mn, 4°C, 4200 rpm), and pellets were gently resuspended in
2 mL of T1 solution (30mM acétate K, 100mm KCI, 10mM CacI2, 15% glycerol and 50mM
MnCI2 added extemporary) and we completed to 15mL. We incubated cells in this solution
for one hour and centrifuge them 10mn at 4°C/ 2300 rpm. We resuspended in 2 mL of solution
T2 (MOPS pH7 10mM, KCI 10mM, CaCI2 75mM, Glycérol 15%) and was aliquoted into
Eppendorf carefully frozen on dry ice with ethanol.

#### Transformation

DNA was resuspended with 10 µL of water and 1 µL of DNA incubated with 50 µL of the
competent cells DH5α on ice for 30 minutes. We realized heat-shock at 42°C for 45s and
transferred quickly tubes on ice for 5mn. We added 950 µL of SOC media per tube, and
incubated them at 37°C for 1 hour shaking at 200-300rpm. We collected 100 µL from each
tubes and spread in triplicate the mixture evenly across the LB agar plates with
chloramphenycol (25 µg/mL). We incubated them with the agar side at the top at 37°C
overnight for approximately 16 hours, because growing with chloramphenycol is slower than
with other antibiotics. The next day we evaluated the number of colonies on a light field based.

#### Plasmid DNA extraction

We used the Monarch Plasmid Miniprep kit from NEB.
We realized all steps of centrifugation at 16000g. 5 mL of culture was pelleted by
centrifugation during 30 seconds and the supernatant was discarded. Cells was totally
resuspended in 200μl of Plasmid Resuspension Buffer (B1) and lysed with 200 μl Plasmid Lysis
Buffer (B2) and 5-6 time inversion tubes. After one minute of incubation and in order to avoid
plasmid irreversible denaturation, we added to neutralize lysate 400 μl of Plasmid
Neutralization Buffer (B3). After 2 minutes, we centrifuge and carefully transfer supernatant
to the spin column and repeat centrifugation for 1mn. We removed RNA, protein and
endotoxin with 200 μl of Plasmid Wash Buffer 1 and then after centrifugation with 400 μl of
Plasmid Wash Buffer2. To finish, we made elution with 30μl of DNA elution buffer. Plasmid
DNA concentration was measured with nanodrop.

#### Polymerisation Chain Reaction

We realized DNA amplification with Q5® High-Fidelity 2X Master Mix (NEB #M0492S). To make
25µL reaction, we added 12.5µL of Q5 High-Fidelity 2X Master Mix, 1.25µL of each forward/
reverse primers (10µM), 250 ng of DNA and we completed with nuclease free water. We
gently mixed reaction and transferred PCR tubes from the ice to a PCR machine with the block
preheated to 98°C and began thermocycling.
We have designed our primers to amplify the desired DNA sequence, adding restriction
recognition sites of the enzymes that will be used in cloning at 5’ ends of the primers, with a
few extra nucleotides prior to the restriction site to increase restriction efficiency. To do so,
we used a Tm calculator tool from  [NEB](https://tmcalculator.neb.com/#!/) to design primer pair
with no more difference of temperature than 5°C.

The thermocycling program was:

* 98°C/ 30s for initial denaturation
* 98°C/ 10s for denaturation, TM°C/ 20s for hybridation, 72°C/ 20s for elongation (X30)
* 72°C/ 2mn for final extension
* hold 4°C

#### Purification products PCR :

We used the Monarch DNA Cleanup extraction Kit from NEB. We realized all steps of
centrifugation at 13K RPM. First, we resuspended PCR products with 80μl of DNA Cleanup
Binding Buffer and transfer mix into spin column. After one minute of spinning we discarded
flow-through. We added 200 μl of DNA Wash Buffer was added before 1 minute of spinning
(these steps were repeated twice time). We transferred columns into a clean 1.5 ml microfuge
tube before to add 20μl of DNA Elution Buffer to the center of the matrix. After 1 minute, we
eluted DNA by one minute of centrifugation. Concentration and purity of DNA was assessed
with nanodrop.

#### Resuspention of synthetized DNA by IDT

DNA was centrifuge 5s/ 300g and TE 1X (1M Tris-HCL pH 7.5-8; 0.5M EDTA pH8; sterile H20)
was added in order to reach a final concentration of 10 ng/uL. Resuspended DNA was vortexed
and incubated 20mn at 50°C before centrifugation 3/5s (300g mini).

#### Cloning and part assembly

We used the Biobrick assembly kit from NEB.

First, we digested purified PCR products with prefix and suffix restriction enzymes. 500 ng of
upstream parts were individually digested with EcoRI and SpeI in a digestion mix containing
5µL of 10X digestion Buffer 2.1, 1 µL of each enzyme and sterile water to complete the mix
until a final volume of 50µL. 500 ng of downstream parts were individually digested by XbaI
and PstI and 100 ng of pSB1C3 plasmid by EcoRI and PstI enzymes in the same conditions that
upstream parts. Digestion mix were incubated at 37°C for 10 minutes and then heat
inactivated at 80°C for 20 minutes. Then, we gather our devices by using the T4 DNA ligase
from NEB. 2 µl of EcoRI/SpeI digested upstream parts were mixed with 2 µl of XbaI/PstI
digested downstream part, 2µL of EcoRI/PstI digested plasmid, 2 µl of 10X T4 DNA ligase
Buffer, 1 µl of ligase and 11 µL of sterile water. Then, ligation mix was incubated at room
temperature for 10 minutes and heat inactivated at 80°C for 20 minutes. The assembly
construction was used for the transformation of E. coli DH5α. After transformation, cells were
spreaded in LB culture overnight in LB+ Chloramphenycol.

#### GFP and RFP quantification:

After transformation, we selected recombinant clones directly by visualization of GFP and RFP
fluorescence through epifluorescent microscope Zeiss Axiovision. We inoculated 4 mL of LB +
Chloramphenycol with colonies emitting GFP and RFP fluorescence and incubated then
overnight at 37°C. The next day, bacterial cells were all diluted in LB+ Chloramphenycol at a
final OD600 of 0,2. We quantified GFP and RFP production of bacteria in 96-well microwell
plate during their exponential stage (OD600 of 0.5). by using the spectrophotometer
Molecular Device SpectraMax M4 with the following set-up.

Transformed cells were synchronized by dilution on LB+ Chloramphenycol at OD600: 0,2.
Cultures were aliquoted (150 µl) into a 96-well plate (Greiner Bio-One) in which OD (600 nm)
and fluorescence for GFP (BBa\_I13500) and RFP (BBa\_I13507) were read using a SpectraMax
M4 fluorescence plate reader (Molecular Devices) along exponential stage.

Finaly, we made ratio between protein synthesis (fluorescence) and growth rate (optical
density) of our different transformed strains and measured each sample in triplicates.

**GFP (BBa\_I13500) :**- Excitation wavelength : 485nm
- Emission wavelenght : 530nm
- Integration time : 500 ms

**RFP (BBa\_I13507) :** - Excitation wavelength : 584 nm
- Emission wavelenght : 607nm
- Integration time : 500 ms

#### SKP cytoplasmic genomic integration:

The DH5a competent strain was transformed with 1uL of the construction and select on
LB+ampicillin (100ug/ml) at 32°C in triplicate. After two days we count few colonies
(approximately 10) resistant to ampicillin. We picked 3 colonies and streaked out once
overnight in LB+ampicillin, 32°C. We started cultures overnight of 3 colonies in LB without
ampicillin at 32°C (this step allows for some loss of plasmid) and grow overnight. We diluted
cultures appropriately to recover single cells (I dilute 10-7 and plate 50 uL of culture) and plate
on LB plates. Grow at 42°C to block replication of the plasmid. Streak colonies once on LB at
42°C to ensure the complete loss of the plasmid.

After this step of integration and before characterization of the effect of SKP in protein
synthesis we made verifications:

A simple checking was to pick the next day 3 colonies to put them in culture in LB+/- ampicillin.

#### Electrophoresis

We realized an agarose gel electrophoresis (1.5%) using agarose powder with 100ml of 1x TAE
buffer, heated until all agarose is dissolved Agar Low Melting… dissolved in TAE 1X before to
add 6μl of Bet. Mix 1μl of 1kbp DNA ladder with 6μl of loading dye (bromophenol blue) and
4μl of 1x TAE buffer (total 6ul) and load onto first well. We Mixed 10μl of PCR products with
2μl of loading dye 6X before loading into wells. Running gel electrophoresis was made at 10V
for 30 minutes approximately and photograph gels under UV light

#### Lysis of bacteria:

To produce the proteins to purify, we used DH5α containing PET21 GST-X10HIS
These bacteria were cultivated from a glycerol stock maintained at -80°C.
Volumes vary depending on the purification system. So, for the sake of clarity the volumes are presented in Table.1.
The lysis of the bacteria took place at the middle of the exponential phase at t = 6h. The experiments were carried out at 4°C. We centrifuged the bacteria at 3000 RPM for 10 min at 4°C. The bacterial pellet was then resuspended in PBS containing lysosyme (1 mg/ml) for 30 min then mechanical lysis was made by vortexing for 10 min. A centrifugation (12000 rpm during 10 min) was then performed and the supernatant was recovered. 60 μL of the supernatant were taken for an SDS-PAGE (unpurified fraction) and a 15 μL fraction was used to study the enzymatic activity.

#### Purification with Gluthation Agarose Resin

From the bacterial lysis supernatant, we purified the GST-10XHIS through a 0.5 ml Glutathione-agarose resin. To do so, we added our sample and centrifuged the resin four times and retrieved the supernatant. Then we added 100µL of GSH 100mM and centrifugated 2times. We recuparated the surpernant which represent the elution sample.
We also took 60 µL and 10 µL of aliquot from it to perform a SDS-PAGE and determine the GST enzymatic activity respectively.

#### Purification HiTrap Column

We used a HiTrap 1ml column to perform this purification.
We used a siringe to file our proteins into the column.
First, we created the binding buffer and the elution buffer.
-Binding buffer (Imidazole 20 mM) : 3.75 ml of Phosphate buffer solution
0.3 ml of Imidazole 2M
qs water to 30 ml
-Elution buffer (Imidazole 500 mM) : 1 ml of Phosphate buffer solution
2 ml of Imidazole 2M
qs water to 8 ml
Before purifying our proteins sample, we preteated it by adding Imidazole so that it has the same concentration as the binding buffer (20 mM).
-Steps for the purification :
1. Fill the syringe with distilled water. Remove the stopper and connect the column to the syringe tubing « drop to drop » to avoid introducing air to the system.
2. Remove the twist-off end.
3. Wash the column with 5 column volumes of distilled water.
4. Equilibrate the column with 5 column volumes of binding buffer with a flow rate of 1ml/min.
5. Apply the pretreated sample.
6. Wash with 5 column volumes of binding buffer.
7. Eluate with 5 column volumes of elution buffer.

#### Mesurement of the GST enzymatic activity

We took aliquots from the supernatant before and after purification in order to measure the enzymatic activity of the GST.
The measurement of this enzymatic activity allows us to obtain a quantitative analysis of the GST and thus a final purification yield.
The measurement of the enzymatic activity of the GST was done through the following reaction:
GST
GSH + CDNB ---> GS-DNB + HCl
Glutathione (GSH) and CDNB are substrates of GST.
The absorbance of GS-DNB at 340 nm allowed us to quantify the enzymatic activity of GST.
Note that the reaction between GSH and CDNB can be done without the presence of GSH. This is the chemical activity. So, we standardized our OD measurements according to this absorbance values.
From this enzymatic activity, a yield can be obtained. Yield = Final total activity / Initial total activity
Initial total activity = Enzyme activity for 5 l \* Total Volume of the supernatant bacterial lysis
Total final activity = Enzymatic activity for 5 l \* purification supernatant Total Volume

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# Team:UPMC PARIS/Experiments

Impact UPMC

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* Manufacturing
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    - [Education](https://2017.igem.org/Team%3AUPMC_PARIS/Education)
    - [Laws and regulations](https://2017.igem.org/Team%3AUPMC_PARIS/Laws_and_regulation)
  + Gold HP
    - [Integrated and Gold](https://2017.igem.org/Team%3AUPMC_PARIS/HP/Gold_Integrated)
    - [Biology](https://2017.igem.org/Team%3AUPMC_PARIS/Gold_HP_Biology)
  + [Public engagement](https://2017.igem.org/Team%3AUPMC_PARIS/Engagement)
* [Awards](#blog)
  + [Entrepreneurship](https://2017.igem.org/Team%3AUPMC_PARIS/Entrepreneurship)
* [Judging form](https://igem.org/2017_Judging_Form?team=UPMC_PARIS)

# Experiments

### Part I - Characterization of the relative strength of some promoters with polycistronism effect

#### Introduction :

The insertion of chaperone genes in bacterial chromosome is an efficient system to ensure
the stability of our optimized E. coli strain for the expression of eukaryotes recombinant
proteins. However, the transposition one hampers system that we used to target a unique
insertion site. To ensure an adequate production of chaperone for the proper folding of all
recombinant proteins, we decided to insert two copies of our chaperone gene through a
polycistronic construct under the control of a strong promoter.

The Anderson Promoters include a large panel of constitutive promoters family with different
levels of strengths. They are mainly including into the J61002 backbone expressing RFP for
color screening. Lot of team projects are based on these types of promoters and they were
previously well characterized. However, their ability to control multiple gene expression was
never determined.

We characterized strength of several promoters from the iGEM community, which are
BBa\_J23101, BBa\_J23102, BBa\_J23104, BBa\_J23107 and BBa\_J23110. In other hand, we
wanted to evaluate their level of strength in the context of polycistronism, with GFP
(BBa\_I13500) and RFP (BBa\_I13507) reporters downstream. We think that it could be very
interesting to assess the ability of these promoters to control several gene expression, in order
to choose which one of them is more appropriate for us to induce an high chaperone
expression.

#### Achievements :

- We documented at least one new substantial contribution to the iGEM community that
showcases a project made with BioBrick.

 - We measured the strength variation of 5 differents promoters in the context of a simple or
multiple gene expression, allowing us to selected for our project the promoter BBa\_J23107,
showing high level of activity.

#### Method

In order to assess the strength of various promoters from the Anderson family we built a
polycistronic reporter including GFP (BBa\_I13500) and RFP (BBa\_I13507) which we places
under the control of characterized promoters.

![](https://static.igem.org/mediawiki/2017/4/46/Upmc_paris_parts1_1.png)

Figure 1 Global strategy about our study of Anderson’s Promoter characterization

### Amplification of iGEM parts

We transformed E. coli DH5α competent bacteria with DNA from the iGEM distribution kit
2017. The Anderson’s promoters BBa\_J23101, BBa\_J23102, BBa\_J23104, BBa\_J23107,
BBa\_J23110, GFP coding sequence BBa\_I13500 and RFP coding sequence BBa\_I13507.

### DNA amplification of parts and cloning of devices

After transformation of bacterial cells we proceed to a plasmid DNA extraction with the the
Monarch Plasmid Miniprep kit from NEB. Then we amplified each part by PCR using the Q5®
High-Fidelity 2X Master Mix (NEB #M0492S) with following primers.

Primer Foward: VF 5’tgccacctgacgtctaagaa 3’

Primer Revers : VF2 5’attaccgcctttgagtgagc 3’ primers

(Tm: 60°C)

We used the Monarch DNA Cleanup extraction Kit from NEB to purify the PCR products.

We used the Biobrick assembly kit from NEB to digest GFP BBa\_I13500 upstream part and RFP
reporter downstream part and cloning the new device GFP-RFP in the pSB1C3 plasmid.

The next day we follow the same scheme in order to assemble our final polycistronic device
Characterized promoter - GFP - RFP.

After transformation, we selected recombinant clones directly by visualization of GFP and RFP
fluorescence through epifluorescent microscope Zeiss Axiovision.

We quantify GFP and RFP production in bacteria in 96-well microwell plate during their
exponential phase (OD600 of 0.5) using the spectrophotometer Molecular Device SpectraMax
M4. Finally, we made ratio between protein synthesis (fluorescence) and growth rate (optical
density) of our different transformed strains and measured each sample in triplicates.

#### Results

![](https://static.igem.org/mediawiki/2017/a/a8/Upmc_paris_parts1_2.png)

![](https://static.igem.org/mediawiki/2017/b/bf/Upmc_paris_parts1_3.png)

Figure 2 Quantification of GFP fluorescence and RFP in the context of polycistronic reporter
under the control of Anderson’s promoters

Promoter strength was assessed through the quantification of specific fluorescence from GFP
(A) and RFP (B) normalized with OD600 to obtain relative fluorescent units (RFU).

#### Discussion

The specific relative fluorescence of the original Anderson Promoters in backbone pSB1C3
shows strength variations with GFP reporter BBa\_I13500. The strongest promoter of the 5
measured ones is BBa\_J23107 (15796,8U) followed by BBa\_J23104 promoter (9443,2 U).
BBa\_J23102 and BBa\_J23110 exhibit a comparable activity than BBa\_J23101 promoter
(approximately 2500 U). Our system of polycistronic reporter show that all of the five
promoters tested allow the a polycistronic production of proteins. Surprisingly, the level of
RFP fluorescence is not affected by the strength of these different promoters. These results
suggest in our study that the level of activity in the context of polycistron is not directly
affected by the strength of the promoter. One hypothesis of these observation is that the
distance between the sequence GFP and RFP was not enough important to look for the effect
on the second reporter expression, and it could be more reliable to use Lux genes in order to
avoid the overlap between fluorescences. To conclude, our work convince us to use
BBa\_J23107 promoter in the second part of our work.

### Part II- SKP integration into bacterial chromosome

#### Introduction :

Our purpose was to produce antibodies through our transportable factory "The BioMaker Factory". As many eukaryotic proteins it depends a lot of various post-traductional modification and a precise tridimensional architecture. In this way, it’s appears important for us to coexpress our recombinant protein with chaperones. However, WHO legislations demand a total safety of sanitary product exempt from any dangerous substance. So il was a necessity for us to avoid usage of antibiotics during cell growth. The direct insertion of chaperone gene within the bacterial genome appeared to be the best way to ensure a stable expression of additional chaperone proteins into the genome of our designed bacteria allowing a proper folding of eukaryotic therapeutical proteins.

#### Method:

To do so, we used pGRG25 integration vector obtained from Addgene in transformed DH5α E. coli. This vector was designed by McKenzie et al to insert DNA sequence in bacterial chromosome without inducing drug resistance of the host. This system uses Tn7 to insert transgenes at a defined neutral site in the chromosome (attTn7). The site is highly conserved and is known to work as a Tn7 attachment site in E. coli and its relatives. The attTn7 sequence is conserved in most (all) bacteria.

#### Construction of cytosolic Skp gene :

We had synthesized a cytosolic form of Skp under the control of T7 promoter and ended by a T7 terminator by IDT.

We amplified by Polymerisation Chain Reaction the Skp CDS preceded by its RBS and ended by the T7 promoter with the following primers, introducing XbaI restriction site and SpeI NotI restrictions sites.

Primer forward : 5’GCGGCCGCTACTAGTATTATTTAACCTG3’ Primer Reverse : 5’CTTCTAGAGCCATGGCTGACAAAA3’

TM :67,3°C

We used the Biobrick Assembly kit to place Skp under the control of the promoter from the iGEM distribution kit 2017 BBa\_J2310 we used this new construct to transformed E. coli competent DH5α.

#### Cloning of Skp in pGRG25 :

Culture of these strain was made at 30°C. Indeed, the plasmid backbone is the easily curable temperature sensitive mutant of pSC101, carrying the pSC101 temperature sensitive origin, which must be grown at 30-32°C to allow replication. So in order to extract plasmids, we inoculate these bacterial cells overnight in culture with ampicillin. We inserted the Skp sequence into the NotI restriction site located on the MCS of pGRG25 and then we transformed E. coli competent DH5α with the cloned vector.

#### Integration of the cytoplasmic form of Skp in E. coli DH5α genome

Cultures of the transformed cloned cells was made overnight without antibiotics at 32°C (this step allows for some loss of plasmid) and then at 42°C to block replication of the plasmid. Colonies were streaked once on LB at 42°C to ensure the complete loss of the plasmid and DNA integration into the genome.

After this step of integration and before characterization of the effect of SKP in protein synthesis we made verifications:

A simple checking was to pick the next day 3 colonies to put them in culture in LB+/- ampicillin.

#### Verification of the genomic insertion of cytosolic Skp

We check out the proper insertion of our Skp by performing a PCR on colonies allowing the specific amplification of cytosolic Skp with the following primers:

Primer forward : 5’GCGGCCGCTACTAGTATTATTTAACCTG3’ Primer Reverse : 5’CTTCTAGAGCCATGGCTGACAAAA3’

TM :67,3°C

Transposition was verified by the absence by PCR amplifying sequences which flank the attTn7 site with the following primers.

Primer Foward 5'GATGCTGGTGGCGAAGCTGT3’ and 5'GATGACGGTTTGTCACATGGA3’

Then, we purified DNA from PCR and we visualized PCR product on an agarose gel electrophoresis (1.5%).

#### Characterization of Skp insertion

In order to assess the effect related to the SKP chromosomique intégration, we transformed our engineered E. coli with our composite part BBa\_I13500-BBa\_I13507 created for the promoter caracterisation under the BBa\_J231107 promoter and then we quantified the production of GFP and RFP.

#### Results Verification of the genomic insertion of cytosolic Skp

After amplification by polymerisation chain reaction of RBS-SKP-Terminateur sequence, which represent a fragment of 622pB we realized a comparison between non integrated DH5α cells and bacterial cells with DNA insertion (at 42°C). Here on these electrophoresis gel we can see that there is a band with a very high level of intensity and some SMIRS. We made a mistake between preparation of PCR and used primers 10 times more concentrated but this observation confirm the fact that we can improve the level of SKP production directly into the genome.

#### Characterization of the SKP insertion:

Promoter strength was assessed through the quantification of specific fluorescence from GFP (A) and RFP (B) normalized with OD600 to obtain relative fluorescent units (RFU).
After transformation we compared the level of GFP and RFP expression between DH5α with and without SKP integration. Firstly, we can find with the same condition of transformation a same range of fluorescence for both reporters compared to the characterisation of Anderson’s promoters. Here we can see that the level of GFP expression (around 20000U) is not significantly change when we introduce SKP into the genome, even if there is a very weak increase in the emission level of GFP and RFP. Our supposition is that GFP is a protein which is relatively simple, which don’t need chaperon proteins or an high level of post traductional modifications, and we didn’t had the time to realize other tests with more complex proteins

### Part III - Determination of the best purification system

#### Introduction

The goal is to test and compare the Gluthation purification system and the hisTrap one to systems to see which one would be the most effective for our box.
To do so, we have purified GST-10XHIS proteins with both of the purification system. We have determined the GST enzymatic activity before and after the purifications to obtain the purification yields. Finally, we have done SDS-PAGE chromatographies to semi-quantify the proteins and compare the results with those obtained with the enzymatic activity.

#### Results

![](https://static.igem.org/mediawiki/2017/6/62/UPMC_team_2017_courbe_DO.png)

Figure.11: Growth curve of the DH5α containing the PET 21 GST-10HIS

All the points data come from a mean done by data collected from 5 cultures. Cultures were incubated in LB at37°C. The DO(600nm) was calculated every hour.

We cultured DH5α containing plasmid pet21 GST-10XHIS. We measured the OD every hour in order to follow the turbidity and thus determine the time corresponding to the exponential phase of growth(fig.11).

#### Analysis of GST enzymatic activity:

We calculated the GST activity of the sample taken before and after the purification. We took 5µL of the aliquotes to do the reaction. We then calculated the total reaction for the all volume before and after lysis that can be found in table.1.
The table.2 presents the the activities and purification yields obtained for both of the purification.

For the HisTrap column, we observe a purification yield equal to 72.1 %.

For the Gluthation Agarose Resin we observe a purification yield equal to 32%.

| Fraction | Enzymatic activity From 5 µL (Δabs/min) | Total enzymatic activity (Δabs/min) | Purification Yield |
| --- | --- | --- | --- |
| F1 HisTrap column | 0.13 | 208 | 72.1% |
| F2 HisTrap column | 0.15 | 150 |
| F1 Gluthation Agarose Resin | 0.75 | 120 | 32% |
| F2 Gluthation Agarose Resin | 0.77 | 38.5 |

Table.2 GST Enzymatic activity obtained and purification yield

#### Analysis of SDS-PAGE gel:

After bacterial lysis, we prepared aliquots from the solutions before and after purification and we performed SDS-PAGE for both types of purification. This is done in order to check the presence of GST-10XHIS and to compare these results with those obtained with the enzymatic analysis.

![](https://static.igem.org/mediawiki/2017/a/af/UPMC_team_2017_analyse_purification.png)

12% polyacrylamide gel. 10 µL deposited with Laemli 1X for each well.
M: Kb Ladder. 1: eluted and purified proteins using a Gluthation-Agarose column. 2: expressed proteins before purification.

![](https://static.igem.org/mediawiki/2017/9/94/UPMC_team_2017_analyse_purification2.png)

Figure.13: SDS-PAGE analyse of purification of GST-10his proteins using a HisTrap column

Before purification, mutliple bands can be seen corresponding to bacterial proteins. However, an intense band at about 27 kDa, which correspond to the GST-10His is observed and suggest a good expression of the protein(fig.12.2). After purification with Gluthation Agarose Resin, we can only see one band corresponding to the GST-10His but smaller than the one seen before purification(fig12.1).

SDS-PAGE of HisTrap column purification : Similarly to what was observed in the previous SDSPAGE, multiple bands and one more intense can be seen at about 27kDa corresponding to the GST-10His, before purification(fig13.2). After purification, only one the GST-10His band is observed at 27kDa. The band looks as intense as the one before purification(fig13.1).

Comparing the results with the SDS-PAGE and the GST enzymatic activity, we can say that the purification yields are consistent with the intensity of the bands observed with the SDS-PAGE. Indeed, with the Gluthation-Agarose column, we calculated a purification yield of 32% which is consistent with the diminution of the band size after purification. Likewise, the purification yield of 73% observed with the HisTrap column is consistent with the size of the band. However, the size of the band after HisTrap purification seems to be the as the one before purifcation so the purification yield should be closer to 100%. We have probably lost GST enzymatic activity during the experiments.

To conclude, the HisTrap column seems to be more efficient at purifing the GST-10His protein than the GST-Trap one. Yet, we should not forget that the HisTrap purification System require buffer containing imidazole. Imidazole is irritating and can cause allergies, so we will have to be even more careful when choosing our following Sephadex exclusion chromaztography to exclude small undesired composants. We should maybe try other types of column like the one capable of binding the SUMO protein for example.
For the box modelisation, we still decided to use the data obtained with the HisTrap column.

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# Team:UPMC PARIS/HP entrepreurship

Impact UPMC

* [Home](https://2017.igem.org/Team%3AUPMC_PARIS)
* Team
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* Project
  + [Description](https://2017.igem.org/Team%3AUPMC_PARIS/Description)
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  + [Parts](https://2017.igem.org/Team%3AUPMC_PARIS/Parts)
* Manufacturing
  + [State of art](https://2017.igem.org/Team%3AUPMC_PARIS/State_of_art)
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* Human practices
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    - [Education](https://2017.igem.org/Team%3AUPMC_PARIS/Education)
    - [Laws and regulations](https://2017.igem.org/Team%3AUPMC_PARIS/Laws_and_regulation)
  + Gold HP
    - [Integrated and Gold](https://2017.igem.org/Team%3AUPMC_PARIS/HP/Gold_Integrated)
    - [Biology](https://2017.igem.org/Team%3AUPMC_PARIS/Gold_HP_Biology)
  + [Public engagement](https://2017.igem.org/Team%3AUPMC_PARIS/Engagement)
* [Awards](#blog)
  + [Entrepreneurship](https://2017.igem.org/Team%3AUPMC_PARIS/Entrepreneurship)
* [Judging form](https://igem.org/2017_Judging_Form?team=UPMC_PARIS)

# Results

Here you can describe the results of your project and your future plans.

##### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

##### You should also describe what your results mean:

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

##### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

##### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

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