iGEM 2016 — Team Peking

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# Team:Peking/Notebook

Standard

* [Home](https://2016.igem.org/Team%3APeking)
* Achievements
  + [Results](https://2016.igem.org/Team%3APeking/Results)
  + [Parts](https://2016.igem.org/Team%3APeking/Basic_Part)
  + [Collaborations](https://2016.igem.org/Team%3APeking/Collaborations)
* Project
  + [Overview](https://2016.igem.org/Team%3APeking/Description)
  + [Design](https://2016.igem.org/Team%3APeking/Design)
  + [Crosslinking](https://2016.igem.org/Team%3APeking/Crosslinking)
  + [Uranyl adsorption](https://2016.igem.org/Team%3APeking/Uranyl-adsorption)
  + [Clearance](https://2016.igem.org/Team%3APeking/Clearance)
  + [Secretion](https://2016.igem.org/Team%3APeking/Secretion)
  + [Final Performance](https://2016.igem.org/Team%3APeking/Demonstrate)
* Modeling
  + [Model of Gel Point](https://2016.igem.org/Team%3APeking/Model/GelPoint)
  + [Model of Mass Distribution](https://2016.igem.org/Team%3APeking/Model/MassDistribution)
  + [Software](https://2016.igem.org/Team%3APeking/Software)
* Practices
  + [Overview](https://2016.igem.org/Team%3APeking/HP/Gold)
  + [Field research](https://2016.igem.org/Team%3APeking/HP/311)
  + [Questionnaire](https://2016.igem.org/Team%3APeking/HP/questionnaire)
  + [Consulting](https://2016.igem.org/Team%3APeking/HP/consulting)
  + [Education & Other](https://2016.igem.org/Team%3APeking/HP/otherHP)
* [Safety](https://2016.igem.org/Team%3APeking/Safety)* Lab
    + [Team](https://2016.igem.org/Team%3APeking/Team)
    + [Attribution](https://2016.igem.org/Team%3APeking/Attributions)
    + [Notebook](https://2016.igem.org/Team%3APeking/Notebook)
  * [Interlab](https://2016.igem.org/Team%3APeking/Interlab)

# Notebook

Thin as it is, the notebook serves as a best gift to look back on our struggle for iGEM.

### Dairy

#### [Week 1 (6/26/2016-7/02/2016)](#collapseOne)

* **Parts Construction:**

+ 3SpyTag (A)assemble
+ SUP PCR
+ pET28a backbonePCR
+ SpyCatcher (B)PCR
+ Monomeric Streptavidin (mSA) PCR

#### [Week 2 (7/03/2016-7/09/2016)](#collapse2)

* **Parts Construction:**

+ His-3A-SUP-pET28a construction
+ His-3A-mSA-pET28a construction
+ His-2B-pET28aconstruction

* **Protein Secretion:**

+ Sequenced the plasmid pBES and confirmed that there are no additional BsaI sites
+ Transformed the plasmid pSB1C3 and extracted it for future use
+ Constructed the signal peptide ImdA on pSB1C3 vector

#### [Week 3 (7/10/2016-7/16/2016)](#collapse3)

* **Parts Construction:**

+ mRFP PCR
+ effoREDPCR
+ YFPPCR

* **Protein Secretion:**

+ Constructed the signal peptide OmpA, LTIIb, PhoA, YjfA, NprE

* **Protein Purification:**

+ BB: 15.440 mg/ml
+ 3A-sup: 9.281 mg/ml
+ 3B (15\*linker): 105.857 mg/ml
+ 3A(15\*linker): fail

* **Uranyl Absorption:**

+ Pre-experiment:We got familiar with the apparatuses in the lab we performed experiments related to uranyl and tried the Arsenazo III method. Finally, we obtained a standard curve of the concentration ranging from 0uM-30uM

#### [Week 4 (7/17/2016-7/23/2016)](#collapse4)

* **Parts Construction:**

+ His-4A-SUP-pET28aconstructione
+ His-6A-SUP-pET28aconstruction
+ His-3A-mRFP-pET28aconstruction

* **Protein Secretion:**

+ Constructed the signal peptide LipA, SacB
+ Constructed the kil protein
+ Tried to construct the OmpA-SUP-pET28a expression plasmid but failed

* **Protein Purification:**

+ 3A(15\*linker): 50.393 mg/ml
+ 3A-sup: 10.25 mg/ml
+ 3A-msa: 10.971 mg/ml
+ His-sup: 6.353 mg/ml

* **Clearance:**

+ We broke the E.coli with ultrasonication, and extracted 3A-mSA protein using a affinity column. Mixed 3A-mSA and 3B in TBS buffer. Then we did a SDS-PAGE electrophoresis and analysis the results ( Fig.1 ).We could find out that 3A-mSA had the ability to crosslink with 3B.

#### [Week 5 (7/24/2016-7/30/2016)](#collapse5)

* **Parts Construction:**

+ His-3A-effoRED-pET28aconstruction
+ His-3A-YFP-pET28aconstruction
+ 3A-SUP-pSB1C3 construction
+ 3A-mSA-pSB1C3 construction

* **Protein Secretion:**

+ Constructed the expression plasmids OmpA-SUP-pET28a and LTIIb-SUP-pET28a and transformed them into BL21(DE3) strain

* **Protein Purification:**

+ 4A-sup: 6.182 mg/ml
+ 6A-sup: 22.320 mg/ml
+ 3A-red: 93.560 mg/ml

* **Clearance:**

+ We tried to measure the reaction capacity of the mSA part of the fusion protein with Biotin-Atto 488 which could show autofluorescence. But results were not useful, because there might be some noncovalent interactions between 3 kDa cutoff centrifuge filters and the molecular.

#### [Week 6 (7/31/2016-8/06/2016)](#collapse6)

* **Parts Construction:**

+ His-4A-mRFP-pET28a construction
+ His-4A-effoRED-pET28a construction
+ His-4A-YFP-pET28a construction
+ His-6A-mRFP-pET28a construction
+ His-6A-effoRED-pET28a construction
+ His-6A-YFP-pET28a construction
+ His-4A-mSA-pET28a construction
+ His-6A-mSA-pET28a construction

* **Protein Secretion:**

+ Constructed additional three signal peptides Bpr, Epr and PelB
+ Tried to construct the expression vector in B.subtilis but all failed

* **Protein Purification:**

+ 3A-yellow: fail
+ 3A-pink: fail
+ 3A-sup: 14.374 mg/ml
+ 3A-msa: 14.262 mg/ml

* **Uranyl Absorption:**

+ We improved our experimental methods and supplemented equipment we needed. In these two days, we reduced our reaction volume from 3mL to 400uL and increased our efficiency. Also, standard curve of concentration ranging from 0uM-30uM was tested.
+ The adsorption capacity of 6A-SUP was tested, even though the data were not parallel, we confirmed that 6A-SUP can absorb uranyl. The highest adsorption rate was 81.56%

* **Clearance:**

+ Last week we got nothing useful. So we changed the protocol about how to use Biotin-Atto 488. We prepared several little affinity columns, immobilized 3A-mSA containing 6x His-Tag, and perfused the columns with solution of Biotin-Atto 488. After measuring the fluorescence intensity, we got a qualitative result that 3A-mSA can react with biotin.

#### [Week 7 (8/07/2016-8/13/2016)](#collapse7)

* **Parts Construction:**

+ 3A-SUP-pSB1C3 construction
+ 3A-mSA-pSB1C3 construction

* **Protein Secretion:**

+ Constructed the expression plasmids of SUP, mSA with different signal peptides: PhoA-SUP-pET28a, PelB-SUP-pET28a, OmpA-mSA-pET28a, LTIIb-mSA-pET28a, PhoA-mSA-pET28a and PelB-mSA-pET28a
+ Constructed the expression plasmids of kil: J23110-J61110-kil-pSB4C5
+ Constructed the expression plasmids of mRFP (E1010), yellow (K592010), eforRed (K592012): OmpA-mRFP-pET28a, LTIIb-mRFP-pET28a, PhoA-mRFP-pET28a, PelB-mRFP-pET28a, OmpA-Yellow-pET28a, LTIIb-Yellow-pET28a, PhoA-Yellow-pET28a, PelB-Yellow-pET28a, OmpA-eforRed-pET28a, LTIIb-eforRed-pET28a, PhoA-eforRed-pET28a and PelB-eforRed-pET28a

* **Protein Purification:**

+ 4A-sup: 15.133 mg/ml
+ His-sup: 30.663 mg/ml

* **Uranyl Absorption:**

+ The adsorption capacity of 3A-SUP and 3A-SUP+3B was tested. We confirmed that 3A-SUP can absorb uranyl, so can 3A-SUP+3B. In today’s experiments, the data was more parallel. We used solution containing uranyl only as control and its uranyl concentration after filtration as the actual concentration of uranyl.

* **Clearance:**

+ To absorb the proteins in the environment, we wanted to use biotin-coated beads. So we bought the reagents and magnetic beads with amino group.

#### [Week 8 (8/14/2016-8/20/2016)](#collapse8)

* **Protein Secretion:**

+ Constructed the expression plasmids of SUP and Spycatcher of different signal peptides for B.subtilis: ImdA-SUP-pBES, NprE-SUP-pBES, SacB-SUP-pBES, YjfA-SUP-pBES, LipA-SUP-pBES, ImdA-Spycatcher-pBES, NprE-Spycatcher-pBES, SacB-Spycatcher-pBES, YjfA-Spycatcher-pBES, LipA-Spycatcher-pBES, Bpr-Spycatcher-pBES

* **Protein Purification:**

+ 3A-msa in the form of inclusion body: 2.273 mg/ml
+ 3A: 29.768 mg/ml

* **Uranyl Absorption:**

+ The adsorption capacity of His-SUP was tested, we confirmed that His-SUP can absorb uranyl and the result was close to the data performed by the author of the paper we referred.
+ We tested the adsorption capacity of 3/4/6A-SUP and 3/4/6A-SUP+3B in one day to exclude unnecessary effects. Then we analyzed the data and found the results were compromising. The capacity were fair and even though the proteins formed colloid, the adsorption capacity barely decreased.

* **Clearance:**

+ Constructed the biotin-coated beads.

#### [Week 9 (8/21/2015-8/27/2015)](#collapse9)

* **Protein Secretion:**

+ Constructed the expression plasmids of Spycatcher, mSA and Red with different signal peptides : PhoA-Spycatcher-pET28a, PelB-Spycatcher-pET28a, LTIIb-Spycatcher-pET28a, ImdA-mSA-pBES, NprE-mSA-pBES, SacB-mSA-pBES, YjfA-mSA-pBES, LipA-mSA-pBES, ImdA-Red-pBES, NprE-Red-pBES, SacB-Red-pBES, YjfA-Red-pBES, LipA-Red-pBES and SUP-pBES without any signal peptide as a control plasmid

* **Protein Purification:**

+ 3A-sup: 20.349 mg/ml

* **Uranyl Absorption:**

+ Water from Weiming lake was collected and simulated sea water was prepared.
+ We tested the adsorption capacity of 3A-SUP+3B in different conditions, including TBS buffer, Weiming lake and simulated sea water.
+ we changed the protein-uranyl ratio from 1:1 to 10:1 to determine whether the adsorption capacity increased.
+ we decreased the uranyl concentration to 5uM.
+ we decreased the uranyl concentration to 13nM and increased the protein-uranyl ratio to 6000:1.

* **Clearance:**

+ Prepared the solution containing 3A protein or 3A-mSA protein, added the beads we made last week, shocked the reaction system adequately for 1h, precipitated the beads with magnetic shelf, and measured the concentration of proteins in the liquid supernatant.

#### [Week 10 (8/28/2015-9/03/2015)](#collapse10)

* **Protein Secretion:**

+ Constructed the expression plasmids of inducible kil with different signal peptides E. coli and SUP with P43 promotor for B.subtilis: T7-Lac promotor-OmpA-SUP, T7-Lac promotor-PhoA-SUP; P43 promotor-ImdA- SUP -PBES; P43 promotor-NprE- SUP -PBES, P43 promotor-SacB- SUP -PBES, P43 promotor-LipA- SUP -PBES, P43 promotor-YjfA- SUP -PBES

* **Secretion examination:**

+ Evaluated the secretion effect for 3ASUP of different signal peptides using western blot.

* **Uranyl Absorption:**

+ We received the ICP-MS results.

* **Clearance:**

+ Prepared to attend CCiC, also known as Central China iGEM Consortium. We had a great week in this meeting, in Sun Yat-Sen University, Guang Zhou, China.

#### [Week 11 (9/04/2015-9/10/2015)](#collapse11)

* **Protein Secretion:**

+ Constructed chromoproteins with the signal peptide, PhoA :
+ PhoA-mRFP-pET28a, PhoA-eforRed-pET28a, PhoA-Yellow-pET28a
+ Handed all the vectors with desired elements to the Test Group for examining the secretion efficiency.

* **Secretion examination:**

+ Evaluated the secretion effect for 3A mSA and 3B of different signal peptides using western blot.

* **Protein Purification:**

+ Purified the cell lysate as well as medium of OmpA SUP using Ni-NTA chromatography.

* **Clearance:**

+ Measured the adsorption capacity of protein network with biotin coated beads. (Crosslinked 3A-mSA and 3B for 1 hour, and then added the beads into the reaction system. The remaining proportion of protein in the environment was measured.

#### [Week 12 (9/11/2015-9/17/2015)](#collapse12)

* **Protein Secretion:**

+ Constructed all the Secretion Parts on pSB1C3 vector and the expression plasmids LBP-pET28a, CBP-pET28a and MBP-pET28a

* **Secretion examination:**

+ Quantified the secretion effect of 3B of different signal peptides using anti-Histag ELISA.

#### [Week 13 (9/18/2015-9/24/2015)](#collapse13)

* **Parts Construction:**

+ 3A-LBP, 3A-MBP and 3A-CBP.

* **Protein Purification:**

+ Purified 3A-SUP, 3A-LBP, 3A-CBP and 3A-MBP. Unfortunately, we didn’t get 3A-MBP due to the damage of chromatography column.

* **Secretion examination:**

+ Quantified the secretion effect of 3B and 3ASUP of different signal peptides using anti-Histag ELISA.

* **Uranyl Absorption:**

+ We repeated experiments on adsorption in different water conditions(boiled and without CO2).
+ We tested 3A-SUP+3B adsorption capacity in TBS buffer with different Ph ranging from 6-9.

* **Clearance:**

+ Prepared the Integrating experiment for the next week.

#### [Week 14 (9/25/2015-10/01/2015)](#collapse14)

* **Secretion examination:**

+ Quantified the secretion effect of 3AmSA and 3ASUP of different signal peptides using anti-Histag ELISA.
+ During this week we also tesedt the influence of IPTG on the secreted concentration. A concentration gradient of IPTG was applied to evaluate the secretion effect of OmpA3B at a time gradient from 1 h to 7 h incubated at 37 ℃.

* **Uranyl Absorption:**

+ We repeated experiments on 3A-SUP+3B adsorption capacity in different water conditions(boiled and without CO2).

* **Clearance:**

+ We prepared the kit to adsorb uranyl from the environment. We used the biotin coated beads to harvest the protein network which had accommodated uranyl of simulate contaminative sea water and fresh water.

### Protocols

[![](https://static.igem.org/mediawiki/2016/0/03/T--Peking--image_protocol_Gel_Extraction.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3AGel_Extraction)

**Gel Extraction**

[![](https://static.igem.org/mediawiki/2016/2/2f/T--Peking--image_protocol_ligation.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Aligation)

**Ligation**

[![](https://static.igem.org/mediawiki/2016/c/c1/T--Peking--image_protocol_temperature_gradient_experiment.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Atemperature_gradient_experiment)

**Temperature Gradient Experiment**

[![](https://static.igem.org/mediawiki/2016/0/09/T--Peking--image_protocol_PCR.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3APCR)

**PCR**

[![](https://static.igem.org/mediawiki/2016/d/d4/T--Peking--image_protocol_integrating_experiment.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Aintegrating_experiments)

**Integrating Experiment**

[![](https://static.igem.org/mediawiki/2016/4/48/T--Peking--image_protocol_purification_of_recombinant_proteins.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Apurification_of_recombinant_proteins)

**Purification of Recombinant Proteins**

[![](https://static.igem.org/mediawiki/2016/5/52/T--Peking--image_protocol_transformation.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Atransformation)

**Transformation**

[![](https://static.igem.org/mediawiki/2016/8/85/T--Peking--image_protocol_testing_adsorption_capacity_of_protein.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Atesting_adsorption_capacity_of_protein)

**Testing Adsorption Capacity of Protein**

[![](https://static.igem.org/mediawiki/2016/5/51/T--Peking--image_protocol_AGE.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3AAGE)

**Agarose Gel Electrophoresis**

[![](https://static.igem.org/mediawiki/2016/e/ed/T--Peking--image_protocol_concentration_gradient_experiment.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Aconcentration_gradient_experiment)

**Concentration Gradient Experiment**

[![](https://static.igem.org/mediawiki/2016/b/b4/T--Peking--image_protocol_biotin-associated_Experiment.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Abiotin-associated_Experiment)

**Biotin-associated Experiment**

[![](https://static.igem.org/mediawiki/2016/d/d4/T--Peking--image_protocol_SDS_Pages.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3ASDS-Pages)

**SDS-Page**

[![](https://static.igem.org/mediawiki/2016/1/10/T--Peking--image_protocol_Western_Blot.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3AWestern_Blot)

**Western Blot**

[![](https://static.igem.org/mediawiki/2016/9/97/T--Peking--image_protocol_pH_Gradient_Experiment.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3ApH_Gradient_Experiment)

**pH Gradient Experiment**

[![](https://static.igem.org/mediawiki/2016/7/76/T--Peking--image_protocol_crosslinking.png)](https://2016.igem.org/Team%3APeking/Notebook/Protocol%3Acrosslinking)

**Crosslinking**