iGEM 2017 — Team Peshawar

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# Team:Peshawar/Notebook

iGEM Peshawar

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  + [Parts](https://2017.igem.org/Team%3APeshawar/Parts)
  + [Basic parts](https://2017.igem.org/Team%3APeshawar/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3APeshawar/Composite_Part)
* [Safety](https://2017.igem.org/Team%3APeshawar/Safety)
* Human Practices
  + [Silver HP](https://2017.igem.org/Team%3APeshawar/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3APeshawar/HP/Gold_Integrated)
  + [Public Engagement](https://2017.igem.org/Team%3APeshawar/Engagement)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3APeshawar/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3APeshawar/Entrepreneurship)
  + [Hardware](https://2017.igem.org/Team%3APeshawar/Hardware)
  + [Measurement](https://2017.igem.org/Team%3APeshawar/Measurement)
  + [Model](https://2017.igem.org/Team%3APeshawar/Model)

## Notebook

Click on each week to expand read about them!

#### Week: 01 5th July – 8th July

![](https://static.igem.org/mediawiki/2017/7/7f/T--Peshawar--NB--1.jpg)

Arrival at IIB at 9 AM
Interactions with the team and our mentors
Toured the department and the lab.
Team divided into two groups for an activity to design a device which will save the egg if
dropped from two storey building.
Sketch our life on charts and then present it.
Received goodie bags.
Met our PI Dr. Faisal. Delivered an inspiring and motivational lecture.
Discussed “Fundamentals of Synthetic Biology”
Social Media 101 Lecture with Mr. Jawad.
Team divided into three groups and tasked to make a video using the tips and apps discussed
during the lecture.

#### Week: 03 17th July – 22nd July

![](https://static.igem.org/mediawiki/2017/e/e7/T--Peshawar--NB--3.jpg)

Lecture on “Tips and Guidelines about iGEM” by Co PI Mr. Adnan Niazi.
Division of labor amongst the team regarding all the aspects and sections of the wiki and beyond.
Presentation of each group about their respective idea.
3 ideas selected via voting which will be going to the next round.
Lit sky lanterns at the end of the day.
Final pitches attended by Dr. Faisal.
Selected “Bio-reporter Fish” as our project idea for iGEM 2017.
Work meet at McDonalds to discuss about the project and its details.
First day in the lab.
Welcomed by Dr. Faisal. Delivered a lecture on lab safety, the traditions and most importantly
the lab honor code.
Lab Safety Training started.
Sarah, our mentor, explained all the basic procedures performed in the lab.
Team divided into two, each performing Miniprep for practice.
Received our DNA Distribution Kit along with a lot of cool goodies.
Team investigated and searched for all the parts required for the project and also its alternatives
to prepare a catalogue.
Learnt about Gel Electrophoresis and performed it.
Performed Classic Miniprep in groups of 3.
Skype with Team Sweden.

#### Week: 05 31st July – 5th August

![](https://static.igem.org/mediawiki/2017/7/77/T--Peshawar--NB--5a.jpg)

Meeting with Dr. Faisal about Human Practices and Outreach.
Discussed about the recently attended Makers Fest event at Lahore. Afterwards discussed
about the deliverables required for medals.
Division of labor called by Dr. Faisal in order to work more efficiently.
Each group prepared a plan for their respective work in form of “Objectives, Activities and
Outcomes”.
Skype session:
1. Team Uppsala. They invited us to join them at their webinar.
Performed MiniPrep using NEB Monarch Kit Method.
Met with Mr. Syed Mahmood Nasir, Inspector General Forestry of Pakistan. Acknowledged our
efforts and also offered his support.
Announcement of KP Summer Camp in the following week.
Different teams will travel to different cities for their outreach sessions.
Preparation for Summer Camp over the weekend.

#### Week: 07 15th August – 19th August

![](https://static.igem.org/mediawiki/2017/d/d3/T--Peshawar--NB--7.jpg)

Optimized the protocol for Restriction Digest.
Discussions:
1. Current progress in lab
2. Team wiki
3. Awards page on wiki
4. Requirements for Gold Medal
5. Blogs &amp; What Ifs
Formulated plans for our Human Practices and what things would we target.
Talked to a few food chains regarding our Human Practices plan.
Meeting with our PI Dr. Faisal today.
Following are things we should look for:
1. Interlab study
2. New functional biobrick
3. Meaningful collaboration
4. Is the project safe, responsible, and good?
5. Input from modeling
6. Optimize new parts
7. Does it work?
Performed:
1. Transformation
2. Competent Cell Prep
3. Antibiotic plates for selection
Quiz about general lab work and problems
Diaries were submitted for checking
Received a Case Study: Elon Musk

---

#### Week: 02 10th July – 15th July

![](https://static.igem.org/mediawiki/2017/c/c3/T--Peshawar--NB--2.jpg)

Lecture with Dr. Sohail on “History of Molecular Biology”
Worked on different ideas for our final project.
Lab diaries received today.
Omar Zeb, a team member gave lecture on Microfluidics.
Skype sessions:
1. Team San Diego
2. Team Mexico
3. Team Pasteur
Brainstorming on ideas.
Team interviews and photo sessions with Mr. Jawad
Team meeting regarding outreach and human practices.
Extensive research about the project and its applications.
Discussion with Dr. Faisal regarding our ideas.
Three-minute pitches in front of Dr. Faisal and a chief guest from San Francisco, USA.
Met with Dr. Nadhman.

#### Week: 04 24th July – 28th July

![](https://static.igem.org/mediawiki/2017/7/7e/T--Peshawar--NB--4.jpg)

Performed:
1. Gel Electrophoresis
2. Restriction Digest
3. PCR
Discussed plans and ideas for unique Human Practices activities.
Discussed ideas about our official logo.
Interaction with a team of medical students from RMC, discussing about SynBio and its impact
and role in the field of health and medicine.
Skype:
1. Team Pittsburg
2. Team Postdam
Four members of the team went to Lahore to attend Makers Fest event.
Rest of the team prepared for outreach session at a local school in Peshawar.
First local outreach session for iGEM Peshawar 2017 at Peshawar Model School, Dalazak Road.
Presentation regarding SynBio followed by DIY DNA Extraction using Kitchen Protocol.
Reading and discussion on biosafety.
Quiz related to our respective perspective on different aspects of our own selves and life.
12 duties in the lab distributed amongst the team.

#### Week: 06 7th August – 11th August

![](https://static.igem.org/mediawiki/2017/e/ea/T--Peshawar--NB--6.jpg)

KP Summer Camp week.
Team travelled 6 divisions in KP in 4 groups.
Combined we visited a total of 11 schools and travelled almost 2075 km, reaching out to 1280+
people in these 5 days.
Activities:
1. SynBio presentation
2. Do It Yourself (DIY) DNA Extraction
3. Interviews
4. Ideas Challenge

#### Week: 08 21st August – 27th August

![](https://static.igem.org/mediawiki/2017/9/9f/T--Peshawar--NB--8.jpg)

Looked for parts in kit plates
1. Part A: RBS + CDS + T (codes yellow color)
2. Part B: Promoter with repressor ArsR
Performed PCR of following things:
1. PCR of
a. Cadmium promoter
b. Arsenic promoter and composite part
c. Ligated products using prefix F suffix R
d. dNTPs to check contamination
e. Colony PCR
2. Media prep for competent cells, Miniprep, broth for culture of Ars cells
3. Gel Electrophoresis
4. Sub-culturing for BioArts
5. R.D
a. Ars Promoter
b. CDS
c. Plasmid
6. Competent Cell Prep
7. Ligations
a. Parts with plasmid in varying concentrations
8. Autoclave
Had a lecture with Dr. Sohail on expression systems and reporters.
Discussed notebook design for wiki
1. Road timeline (ushma)
2. Coral reef design (Dawood)
Samples of RD were run on gel. All results were positive.
Prepared transformations for inter-lab study.
Cells then plated on petri plates
Transformations for inter lab were checked. We got positive results as well.
Results of competent cells after 17 hours were observed.
Results of PCR not satisfactory, now moving forward with broth miniprep and then RD.
Troubleshooting of ligation carried out

---

#### Week: 09 28th August – 29th August

![](https://static.igem.org/mediawiki/2017/e/e5/T--Peshawar--NB--9.jpg)

Performed
1. Digestion of miniprep of Arsenic device.
2. Gel Electrophoresis of R.D products.
3. Prepared samples for interlab study.
4. Prepared Master Mix for PCR
Meeting with Dr. Faisal regarding interlab study and human practices.
Logo designs discussed by the team.
Two members of the team went to NUST, Islamabad to complete Interlab study.
Meeting with Dr. Faisal, discussed about the informative booklet plan and the what if campaign.

#### Week: 11 11th September – 16th September

![](https://static.igem.org/mediawiki/2017/5/57/T--Peshawar--NB--11.jpg)

Performed:
1. Transformation was done using the same protocol but a few minute changes:
a. 10 ul DNAse fresh water in plate for resuspension
b. DNA sample 1.0 ul instead of 0.5 ul
c. 300 ul SOC media instead of 400 ul.
2. Ligations
3. Miniprep
4. Gel Electrophoresis
5. Restriction Digest
a. Hg device
b. Cd Promoter
c. YFP
d. D.D of RFP
All plates showed positive results.
Gel Electrophoresis to check results.
Work on Sponsorship Proposal also started.
Received package from Team in Taiwan for testing their device.
Study about mercury chloride salt solution preparation to test our Hg device.

#### Week: 13 25th September – 30th September

![](https://static.igem.org/mediawiki/2017/d/d6/T--Peshawar--NB--13.jpg)

Performed:
1. Ligations:
a. Hg promoter and YFP
i. With plasmid
ii. Without plasmid

2. PCR:
a. Hg-Hg ligated product
b. Colony PCR
i. RFP 2

3. Media preparation for salt testing
4. Salt solution preparation
Checked OD of broths with different ppm salts.
Gel Electrophoresis

#### Week: 15 9th October – 14th October

![](https://static.igem.org/mediawiki/2017/d/dc/T--Peshawar--NB--15.jpg)

Tasks specified to three members in the lab by Dr. Faisal.
1. Arsenic device working ppm literature
2. Cd device basic part ligation
3. IDDP Theory
Performed:
1. PCR
a. 1N (RBS) with pSB1C3
2. Restriction Digest
a. S.D of pCadA and RBS
b. D.D of pSB1C3
c. D.D of 7G (pCadA) and 1N (RBS)
3. Ligation
a. 7G (pCadA) with 1N (RBS)

Gel Electrophoresis
iGEM Peshawar 2017 Survey is online.
Talk session at Rehman Medical College (RMC) on Synbio and applications in health and
medicine.

#### Week: 17 23rd October – 28th October

![](https://static.igem.org/mediawiki/2017/5/5a/T--Peshawar--NB--17.jpg)

Performed:
1. Competent cell preparation
2. Ligation
a. 10O + 6P
b. 10O + 6P\*
c. 10O + 7J
d. 10O + 7J\*
e. IDT parts (merR, Lilac CDS, PnsrB) with pSB1C3
3. Transformations
Fish Farm visit to discuss about our integrated human practices
Part submission DONE!

#### Week: 10 7th September – 9th September

![](https://static.igem.org/mediawiki/2017/0/00/T--Peshawar--NB--10.jpg)

Performed:
1. Antibiotic media preparation
2. Gel Electrophoresis
3. R.D of pSB1C3Prepared antibiotic media
Meeting with Dr. Faisal regarding:
1. Questionnaire designed to analyze people’s response towards SynBio products
2. What Ifs campaign
3. Updates about current progress and future line of action regarding project
4. Need to read some articles

#### Week: 12 18th September – 23rd September

![](https://static.igem.org/mediawiki/2017/b/b5/T--Peshawar--NB--12.jpg)

Performed:
1. R.D
a. Cd device
b. RFP
c. 3G
d. RFP 2
2. PCR
3. Ligations:
a. RFP
b. 3O + YFP
c. RFP 2 + pSB1C3
d. Cd device
Gel Electrophoresis

#### Week: 14 2nd October – 7th October

![](https://static.igem.org/mediawiki/2017/0/0a/T--Peshawar--NB--14.jpg)

Performed:
1. Restriction Digestions
a. P2 ligation device
b. 1O (Hg device)
c. YFP
d. RFP 2 Miniprep
e. 3F (terminator)
2. Ligations
a. 1O + YFP + pSB1C3
b. 1O + YFP
3. PCR
a. 1O + YFP
i. With Taq
ii. With Q5
b. RFP 2
i. With Taq
ii. With Q5

Gel Electrophoresis
Meeting with Dr. Faisal about the deliverables, what if cards and general survey.
Redistribution of tasks amongst the available team members.

#### Week: 16 16th October – 21st October

![](https://static.igem.org/mediawiki/2017/0/06/T--Peshawar--NB--16.jpg)

Report to Dr. Faisal on content for wiki.
Performed:
1. Restriction Digest
a. D.D of composite part 6P (RBS + YFP + TT)
b. D.D of 10O and 6P
c. D.D of 7G (pCadA), 6P and 7J
2. PCR
a. 7G (pCadA)
b. Ligation products
i. 10O + 6P + pSB1C3
ii. 10O + 7J + pSB1C3

3. Ligation
a. 10O + 6P
b. 10O + 7J
c. 10O + 6P + pSB1C3
d. 10O + 7J + pSB1C3
4. Transformations
Prove IDDP Theory by checking O.D every 30 minutes. RFP 2 expresses color first.
Gel Electrophoresis
Discussions
1. IDDP Theory
a. Graphs
b. Tables
2. Part Improvement
3. Project Improvement
4. Protocols page for wiki.

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* iib@cecos.edu.pk
* Institute of Integrative Biosciences,

  CECOS University of IT & Emerging Sciences,

  Phase 6, F-5, Hayatabad, Peshawar, Pakistan
* +92 91 5860291

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# Team:Peshawar/Experiments

iGEM Peshawar

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* Parts
  + [Parts](https://2017.igem.org/Team%3APeshawar/Parts)
  + [Basic parts](https://2017.igem.org/Team%3APeshawar/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3APeshawar/Composite_Part)
* [Safety](https://2017.igem.org/Team%3APeshawar/Safety)
* Human Practices
  + [Silver HP](https://2017.igem.org/Team%3APeshawar/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3APeshawar/HP/Gold_Integrated)
  + [Public Engagement](https://2017.igem.org/Team%3APeshawar/Engagement)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3APeshawar/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3APeshawar/Entrepreneurship)
  + [Hardware](https://2017.igem.org/Team%3APeshawar/Hardware)
  + [Measurement](https://2017.igem.org/Team%3APeshawar/Measurement)
  + [Model](https://2017.igem.org/Team%3APeshawar/Model)

## Experiments

Our Lab Protocols

####

#### AGAROSE GEL PREPARATION

Things required:

1. 1X TAE Buffer
2. Agarose
3. EtBr

Protocol:

Calculate the amount of agarose required for 50 ml TAE Buffer based on percentage of gel required.

2 % Agarose Gel:

1. 50 ml 1X TAE Bufeer
2. 1.0 g Agarose
3. 0.5 ul EtBr

Mix thoroughly and then pour in gel caster and attach comb. Let it solidify for 30 – 40 minutes.

#### MEDIA PREPARATION

Things required:

1. Nuclease Free Water
2. Yeast
3. Tryptone
4. NaCl
5. Agar

100ml Broth Preparation:

1. NF Water                                100ml
2. Yeast                                       0.5 g
3. Tryptone                                  1.0 g
4. NaCl                                        1.0 g

Mix all these in a beaker and autoclave for 20 minutes at 121OC and 15 psi.

100ml Agar Preparation:

1. NF Water                                100 ml
2. Yeast                                       0.5 g
3. Tryptone                                  1.0 g
4. NaCl                                        1.0 g
5. Agar                                        1.0 g

Mix all these in a beaker and autoclave for 20 minutes at 121OC and 15 psi.

#### ANTIBIOTIC SOLUTIONS

|  |  |  |  |
| --- | --- | --- | --- |
| **ANTIBIOTIC** | **STOCK SOLUTION** | **WORKING SOLUTION** | **SOLUTION MAKING** |
| amp | 100 mg/ml | 100 ug/ml | ddH2O |
| chl | 25 mg/ml | 25 ug/ml | EtOH |
| ken | 50 mg/ml | 50 ug/ml | dH2O/ddH2O |
| tet \*light sensitive\* | 10 mg/ml | 10 ug/ml | dH2O/ddH2O |

#### CLASSIC MINIPREP

Protocol:

1. Resuspend E. coli / Plasmid cells by shaking
2. Label 2 1.5 ml Eppendorf tubes.
3. Transfer 1000ul of E. coli / Plasmid (overnight suspension)
4. Close caps – then spin for 1 minute to pellet the cells.
5. Discard supernatant – drain off completely.
6. Add 100ul of ice-cold GTE solution to each tube – resuspend by pipetting up and down till it is homogeneous.
7. Add 200 ul of SDS/NaOH solution to each tube – close caps – mix rapidly inverting tubes 5 times.
8. Stand tubes on ice for 5 minutes.
9. Add 150 ul of ice-cold KOA solution – close caps – mix rapidly inverting 5 times. (precipitate will be made)
10. Stand tubes on ice for 5 minutes.
11. Spin for 5 minutes to pellet the precipitate.
12. Transfer 400 ul of supernatant from each tube into clean 1.5 ml Eppendorf tubes.
13. Add 400 ul of isopropanol to each tube of supernatant – close cap – invert 5 times.
14. Stand at room temperature for only 2 minutes.
15. Spin for 5 minutes.
16. Discard supernatant.
17. Add 200 ul of 100% ethanol.

---

####

#### MINIPREP – NEB MONARCH KIT METHOD

Things required:

1. Sample culture
2. Resuspension buffer (B1)
3. Lysis buffer (B2)
4. Neutralization buffer (B3)
5. Wash buffer 1
6. Wash buffer 2
7. Elution buffer

Protocol:

1. Take 5 ml of culture in an Eppendorf tube.
   1. Centrifuge at 13000 rpm for 30 seconds.
   2. Take pellet.
   3. Repeat numerous times for desired amount of culture.
   4. Mix 200 ul Resuspension buffer (B1) using vortex or pipette.
   5. Add 200 ul of Lysis buffer (B2) and gently invert 5 – 6 times, then incubate at room temperature for 1 minute. Color should change to purplish pink. DO NOT VORTEX.
   6. Add 400 ul of Neutralization buffer (B3) and gently invert till the color changes to yellow. Precipitate will be formed. Incubate at room temperature for 2 minutes.
   7. Centrifuge at 13000 rpm for 5 minutes.
   8. Collect supernatant in the column provided with the kit.
   9. Centrifuge for 1 minute, and discard the flow through.
   10. Add 200 ul Wash buffer 1 and then centrifuge for 1 minute. Discard the flow through.
   11. Add 400 ul Wash buffer 2 and then centrifuge for 1 minute. Discard the flow through.
   12. Transfer the contents of the flow through to a clean 1.5 ml Eppendorf tube.
   13. Add 30 ul Elution buffer.
       1. Incubate for 1 minute at room temperature.
       2. Centrifuge for 1 minute.
       3. Discard the column.
       4. Mark the Eppendorf tube and place the plasmid DNA at -20OC for further use.

#### POLYMERASE CHAIN REACTION

Reaction volume: 25 ul

Three main steps:

1. Initial Denaturation:                            95OC for 5 minutes
   1. Denaturation:                          95OC for 30 seconds
   2. Annealing:                              65OC for 15 seconds
   3. Extension:                               68OC for 30 seconds – 90 seconds (depends on size)
   4. Final Extension:                                  68OC for 5 minutes

DNA Polymerase Types:

1. Taq                                                      1 x
2. Pyrococcus furiosus                            6 x
3. Phusian DNA Polymerase                  50 x
4. Q5

Protocol:

1. Standard Rxn Buffer                          2.5 ul
2. dNTPs                                                 0.5 ul
3. Forward Primer                                   0.5 ul
4. Reverse Primer                                    0.5 ul
5. Template DNA                                   1-2 ul
6. Polymerase Enzyme                           0.125 ul
7. Nuclease Free Water                           upto 25 ul

#### COLONY POLYMERASE CHAIN REACTION

*For colony PCR, take a colony of choice from a plate and mix in 10 ul of NF water. Then take    1 - 2 ul of sample from that mixture*

Reaction volume: 25 ul

Three main steps:

1. Initial Denaturation:                            95OC for 5 minutes
   1. Denaturation:                          95OC for 30 seconds
   2. Annealing:                              65OC for 15 seconds
   3. Extension:                               68OC for 30 seconds – 90 seconds (depends on size)
   4. Final Extension:                                  68OC for 5 minutes

DNA Polymerase Types:

1. Taq                                                      1 x
2. Pyrococcus furiosus                            6 x
3. Phusian DNA Polymerase                  50 x
4. Q5

Protocol:

1. Standard Rxn Buffer                          2.5 ul
2. dNTPs                                                 0.5 ul
3. Forward Primer                                   0.5 ul
4. Reverse Primer                                    0.5 ul
5. Template DNA                                   1-2 ul
6. Polymerase Enzyme                           0.125 ul
7. Nuclease Free Water                           upto 25 ul

#### RESTRICTION DIGESTION

Reaction volume: 50 ul

1. SINGLE DIGESTION
   1. Buffer (Cut Smart or 3.1)                               5 ul
   2. Restriction Enzyme                                         1 ul
   3. DNA Sample                                                   1 ul
   4. Nuclease Free Water                                       upto 50 ul

1. DOUBLE DIGESTION
   1. Buffer (Cut Smart or 3.1)                               6 ul
   2. Restriction Enzyme                                         1+1 ul
   3. DNA Sample                                                   2 ul
   4. Nuclease Free Water                                       upto 50 ul

PROCEDURE:

1. Mix all components in 1.5 ml Eppendorf tube
2. Place the tube at 37OC for 60 minutes
3. Then place the tube at 80OC for 20 minutes. This step is called as heat-kill step.
4. Store the tube at -20OC for further use.

---

#### RESTRICTION DIGESTION (OPTIMIZED)

Reaction volume: 10 ul

1. Single Digestion
   1. Buffer                                                             2.5 ul
   2. Restriction Enzyme                                         1 ul
   3. DNA Sample                                                   3-6 ul (depends on conc. of sample)
   4. Nuclease Free Water                                       upto 10 ul
   5. BSA (if using buffer 2)                                   0.5 ul

1. Double Digestion
   1. Buffer                                                             4 ul
   2. Restriction Enzyme                                         1+1 ul
   3. DNA Sample                                                   4-6 ul (depends on conc. of sample)
   4. Nuclease Free Water                                       upto 10 ul
   5. BSA (if using buffer 2)                                   0.5 ul

Procedure:

1. Mix all components in 1.5 ml Eppendorf tube
2. Place the tube at 37OC for 60 minutes
3. Then place the tube at 80OC for 20 minutes. This step is called as heat-kill step.
4. Store the tube at -20OC for further use.

#### LIGATION (NEB)

Things Required:

1. Buffer (NEB)
2. Enzyme
3. Insert (Part A, Part B, or both)
4. Plasmid Backbone/ Vector
5. Nuclease Free Water

Protocol:

1. Take 4 ul Buffer in a small tube.
2. Add Insert and Vector in 3:1 molar concentration values.
3. Add NF water upto 19 ul.
4. Add 1 ul Ligase enzyme.

Place the tube in Thermal Cycler at 16OC for 60 minutes.

Place it at 80OC for 20 minutes for heat kill.

Mark the tube and store at -20OC for further use.

#### LIGATION (INVITROGEN)

Things Required:

1. Buffer (Invitrogen)
2. Enzyme
3. Insert (Part A, Part B, or both)
4. Plasmid Backbone/ Vector
5. Nuclease Free Water

Protocol:

1. Take 4 ul Buffer in a small tube.
2. Add Insert and Vector in 3:1 molar concentration values.
3. Add NF water upto 19 ul.
4. Add 1 ul Ligase enzyme.

Place the tube in Thermal Cycler at 25OC for 60 minutes.

Place it at 65OC for 10 minutes for heat kill.

Mark the tube and store at -20OC for further use.

#### COMPETENT CELL PREPARATION

Protocol:

1. Inoculate 0.2 ml (200 ul) of overnight culture in 10 ml LB broth.
2. Put in shaker incubator at 37OC for 2 hours.
3. Check O.D 600. It should be 0.2 – 0.3
4. Take a centrifuge tube and place it on ice to cool.
5. Place the culture on ice for 5 minutes
6. Transfer it to pre – cool centrifuge tube
7. Centrifuge at 6000 rpm for 5 minutes at 4OC.
   1. Discard supernatant
   2. Gently resuspend the cells with ice – cold 50 Mm CaCl2
   3. Leave on ice for 40 minutes.
   4. Centrifuge at 6000 rpm for 5 minutes at 4OC.
      1. Discard supernatant.
      2. Gently resuspend with 2 ml CaCl2
      3. Store on ice until needed.
      4. Make aliquots of 100 ul and place at -80OC.

#### TRANSFORMATION

Things Required:

1. Competent cells (DH5a)         100 ul
2. Plasmid DNA                         1 ul
3. SOC Media (NEB)                 400 ul

Protocol:

1. Thaw the competent cells on ice (-80OC to 4OC)
2. Add 100 ul of C.C in 1.5 ml Eppendorf tube.
3. Mix 1 ul of DNA in100 ul of C.C
4. Incubate on ice for 40 minutes at 4OC.
5. Give the cells a heat shock for 60 seconds (4OC to 42OC). This is a very critical step.
6. Incubate on ice for 5 minutes.
7. Add 400 ul of SOC media in LFH.
8. Incubate the mixture in incu-shaker at 37OC for two (2) hours.
9. Plate the cells.
10. Wrap with parafilm and incubate in incubator at 37OC overnight.

* [@iGEM Peshawar](https://www.facebook.com/iGEMPeshawar/)
* [@iGEM Peshawar](https://twitter.com/igem_peshawar)
* [@iGEM Peshawar](https://www.instagram.com/igempeshawar/)

* iib@cecos.edu.pk
* Institute of Integrative Biosciences,

  CECOS University of IT & Emerging Sciences,

  Phase 6, F-5, Hayatabad, Peshawar, Pakistan
* +92 91 5860291

[![](https://static.igem.org/mediawiki/2017/f/f7/T--Peshawar--iib-logo-small-compressed.png)](http://iib.cecos.edu.pk/)
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# Team:Peshawar/Results

iGEM Peshawar

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[![iGEM Peshawar](https://static.igem.org/mediawiki/2017/4/4e/T--Peshawar--Team-Logo.png)](https://2017.igem.org/Team%3APeshawar)

* [Home](https://2017.igem.org/Team%3APeshawar)
* Team
  + [Team](https://2017.igem.org/Team%3APeshawar/Team)
  + [Collaborations](https://2017.igem.org/Team%3APeshawar/Collaborations)
* Project
  + [Description](https://2017.igem.org/Team%3APeshawar/Description)
  + [Design](https://2017.igem.org/Team%3APeshawar/Design)
  + [Experiments](https://2017.igem.org/Team%3APeshawar/Experiments)
  + [Notebook](https://2017.igem.org/Team%3APeshawar/Notebook)
  + [Interlab](https://2017.igem.org/Team%3APeshawar/InterLab)
  + [Contributions](https://2017.igem.org/Team%3APeshawar/Contribution)
  + [Model](https://2017.igem.org/Team%3APeshawar/Model)
  + [Result](https://2017.igem.org/Team%3APeshawar/Results)
  + [Improve](https://2017.igem.org/Team%3APeshawar/Improve)
  + [Attributions](https://2017.igem.org/Team%3APeshawar/Attributions)
* Parts
  + [Parts](https://2017.igem.org/Team%3APeshawar/Parts)
  + [Basic parts](https://2017.igem.org/Team%3APeshawar/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3APeshawar/Composite_Part)
* [Safety](https://2017.igem.org/Team%3APeshawar/Safety)
* Human Practices
  + [Silver HP](https://2017.igem.org/Team%3APeshawar/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3APeshawar/HP/Gold_Integrated)
  + [Public Engagement](https://2017.igem.org/Team%3APeshawar/Engagement)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3APeshawar/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3APeshawar/Entrepreneurship)
  + [Hardware](https://2017.igem.org/Team%3APeshawar/Hardware)
  + [Measurement](https://2017.igem.org/Team%3APeshawar/Measurement)
  + [Model](https://2017.igem.org/Team%3APeshawar/Model)

## VALIDATED PART

Our team has successfully validated a newly designed RFP coding device (RFP2) that contains double RFP reporting system. Our designing and wet lab experiments not only proved that our part has properly and efficiently worked but also helped us improve one of the most used parts in the iGEM registry, BBa\_J04450.

Results are vivid; our validated part that we named RFP2 is showing brighter fluorescence under UV light and giving more intense color (purplish-red) under visible light than the random RFP device.

![](https://static.igem.org/mediawiki/2017/e/ea/T--Peshawar--Improve-1.png)

RFP2 under UV light along with controls, as brighter and beautiful as neon!

![](https://static.igem.org/mediawiki/2017/a/a7/T--Peshawar--Results-Last-1.jpg)

RFP2 under visible light, shocking pink and yummy!

We successfully improved one of the most frequently used parts in iGEM; RFP coding device BBa\_J04450. The idea itself was based on our IDDP theory; ligating 2 RFP devices together and inserting them both in a single plasmid might make the color more intense and decrease the expression time to a certain extent.

![](https://static.igem.org/mediawiki/2017/9/93/T--Peshawar--Improve-2.png)

Above gel shows the bands of our miniprep-ed RFP2 plasmids that were directly transformed from the ligated product. As it was direct transformation, some of the self-ligated plasmids too were extracted.

**Check out our [IDDP theory](https://2017.igem.org/Team%3APeshawar/Model) for further understanding!**

For the projects like us in which a biosensor gives color indication upon interacting with a target molecule, this **improvement** holds massive importance. By decreasing the expression time one can detect target compounds much earlier.

Our engineered bacteria that contain double-RFP expressing plasmid showed visible difference in the color as well as fluorescence in just 5hours of incubation. Plasmid having single-RFP device started to change the color after seventeen hours!

![](https://static.igem.org/mediawiki/2017/7/7a/T--Peshawar--Improve-3.png)

The picture above further fortifies the **improvement** that we have done. Sub-cultures of the transformed colonies clearly differentiate RFP and RFP2; our improved part.

* [@iGEM Peshawar](https://www.facebook.com/iGEMPeshawar/)
* [@iGEM Peshawar](https://twitter.com/igem_peshawar)
* [@iGEM Peshawar](https://www.instagram.com/igempeshawar/)

* iib@cecos.edu.pk
* Institute of Integrative Biosciences,

  CECOS University of IT & Emerging Sciences,

  Phase 6, F-5, Hayatabad, Peshawar, Pakistan
* +92 91 5860291

[![](https://static.igem.org/mediawiki/2017/f/f7/T--Peshawar--iib-logo-small-compressed.png)](http://iib.cecos.edu.pk/)
[![](https://static.igem.org/mediawiki/2017/2/23/T--Peshawar--govt-of-kpk-logo-small-compressed.png)](http://kp.gov.pk/)
[![](https://static.igem.org/mediawiki/2017/f/f0/T--peshawar--dost-logo-small-compresed2.png)](http://www.dost.gov.pk/)
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