Experiments

Plan

We have a lot of work to do on our project regarding the wet lab. This work needs to be regulated and well planned, so we divided it into 3 main phases. Each phase will contain a group of experiments that relate to each other to get the results that will be used in the next step:

• Phase 1 : Safety Practice
  Before starting our wet lab phase, the wet lab team must refresh their information about lab safety and technique to keep them safe during this phase.

Phase 2 : Cell Lab
This step will focus on cell culture and growth to produce the mature cells that will be ready to be transfected.

• Phase 4 : Validation
  It concerns the part of DNA from resuspension , digestion, ligation, and gel electrophoresis.

Phase 3 : Molecular lab
This phase concerns validation of the expression of the transfected part with specific antibodies and tagging it with specific tagging for each antibody.

Phase 1: Safety practice

During this phase, the wet lab team went to the lab to revise the general safety rule to prevent any disaster from happening during the wet lab work. Also, they were trained in the basic lab techniques to improve the efficacy of the work, prevent material and time waste, and reduce mistakes resulting from a lack of experience.

Day 1: Identification of safety signs and lab equipments

Recorder: Omar Fouad
Attendance: All team members

Firstly, we have learned that missing a single procedure, such as labelling the products, can ruin months and years of work. Either in clinical or biological labs, labels are crucial and can be variable according to your needs. Furthermore, they can be used for:


• Organization: you can organize your products, materials,or equipment by names, colors,date of expiration,or even barcodes. Printed IDs or barcodes help you know where the item is, what steps it has undergone,and what steps are left.
  

• Accuracy: The easier you can collect your data and work, the more accurate your results are.
  

• Safety: Any mistake in labels can lead to a catastrophe. For example, mixing incompatible agents can result in unpredictable reactions or, clinically, replace patient results.
  

Similarly, the safety signs have the same importance. We have learned some signs related to our laboratory work, such as no food or drinks permitted, personal protective equipment related to our biosafety level, biohazard signs, and carcinogenic signs.

Moreover, we have learned how to use some of the laboratory devices , such as PCR, centrifuges, etc…

Day 3: Safe by practice

Recorder: Ahmed Wael
Attendance: All team members
Finally, we started to learn how to use some equipment properly under the supervision of experts. In addition, how to irrigate the eyes or any body part if exposed to corrosive substances. As medical students, we also know how to use the first aid box and how to prevent further complications.

Phase 2: Molecular lab

During the molecular lab phase, we aim to prepare the plasmids carrying our genetic circuits for transfection in the cell lab phase. We started our molecular phase by resuspension of the dried genetic parts ordered from IDT. Then it was digested by the restriction enzymes mentioned below. After that, we transform the plasmid vector pcDNA 3.1- into bacteria for amplification to prepare the proper amount of the plasmid vector to carry our circuits. Then the genetic circuits were ligated with the plasmid vector to form our plasmids. In addition to that, we have run gel electrophoresis after digestion and ligation to be sure about the length of the plasmid vector and the genetic parts and to ensure that they were added to the right place.

Preservation of pcDNA3.1(-)

DH5α competent cells preparation-chemical methods

Recorder: Omar Fouad
Attendance: Hossam algamal , Ahmed wael and Mohamed Aboelghar
Documentation of the day:
Location: AFCM Lab

• Today's experiment in the lab focused on preparation of the DH5α competent cells to be ready for transformation.

• Our goal today is to prepare DH5α competent cells to transform them with pcDNA 3.1-.

• We started our experiments by adding the overnight cultured media to 500 ml of SOC medium in a 2 L flask.

• Then we let the cell grow to an OD600 between 0.4 or 0.6.

• Then we cooled the cells for 10 min.

• Then we transfer the culture to a sterile 500 mL centrifuge tube then we centrifuge them at a sterile 500 mL.

• Then we resuspend the cells into 150 mL of cold CMG buffer.

• Then we incubated them on ice for 15 min.

• Then We centrifuged the precipitated cells again at 4000 g for 5 min at 4 °C.

• After that we resuspended the cells in 36 mL of cold CMB buffer and transferred them to a 50 mL disposable centrifuge tube.

• We added 1.26 mL of high quality DMSO again, and mixed well, then incubated them on ice for 5 min.

• Then we incubated the cells’ suspension on ice for 5 min.

• We added 1.26 mL of high quality DMSO again, and mixed well, then incubated them on ice for 5 min.

• Then we dispensed the suspension into sterile microcentrifuge tubes and we froze them in a liquid nitrogen.

• Finally we stored the cells at -80.

pcDNA transformation into DH5α and culture on ampicillin plates

Recorder: Ahmed Wael
Attendance: Hossam algamal , Ahmed wael , Mohamed Aboelghar
Documentation of the day:
Location: AFCM Lab

• We removed the cells from the -80 C freezer and thawed in your hands.

• We added 1-5 µl containing 1 pg-100 ng of pcDNA 3.1 to the cells and carefully flicked 4-5 times.

• We placed the mixture on ice for 2 min.

• We heated it at 42 C for 30 sec.

• We placed it on ice for another 2 min.

• We added 950 µl of room temperature SOC to the mixture, then immediately spreaded 50-100 µl onto a selection plate and incubated overnight at 37-42°C.

Alkaline lysis miniprep

Recorder: Mohamed Aboelghar
Attendance: Hossam algamal , Ahmed wael , Mohamed Aboelghar
Documentation of the day:
Location: AFCM Lab

• We prepared the following solution:
  

  • Solution-1:

    • 25 mM Tris-HCl (pH 8)

    • 50 mM glucose

    • 10 mM EDTA

    *Stored at 4 C

  • Solution-2: Denaturing buffer

    • 0.2 N NaOH

    • 1.0% SDS

    *Stored at room temperature

  • Solution-3: Renaturing buffer

    • 120 mL 5M Potassium acetate

    • 23 mL glacial acetic acid

    • 57 mL of dH2O

    *Stored at 4 C

• We cultured 1.5 ml of the overnight bacteria Then we added to the 1.75 ml centrifuge tube.

• We centrifuged the tube at 10,000 g for 30 ml.

• We removed the supernatant and resuspended the bacteria in 100 μL of the resuspension buffer.

• We vortexed the solution for 2 min until the bacteria were fully resuspended

• We added 200 μL of the denaturing solution to the resuspended bacteria, and we inverted 5 times to mix the content.

• We incubated the solution on ice for 5 min.

• We added 150 μL of the renaturing solution to the denatured bacteria, then we mixed it by inverting the tube several times.

• We incubated the solution on ice for 5 min.

• We centrifuged the solution at 12,000 g for 5 min.

• We pipetted the supernatant into a new tube.

• We added 700 μL of cold 100% ethanol to the solution.

• We poured out the supernatant.

• We washed the solution with 70% ethanol to remove excess salt.

• We dried the pellet on air for 20-30 min

• We resuspend the pellet with 25-50 μL of TE.

Resuspension

Recorder: Khalid Hela
Attendance: Hossam algamal , Omar Fouad , Emad Hamdy
Location: AFCM Lab
Documentation of the Day:

• Today we aim to work on our gBlocks Gene Fragments Under the guidance of Dr. Ahmed, the expert in molecular biology,as we aim to resuspend the anticipated gBlocks Gene Fragments from IDT.

• The gBlocks contain genetic material waiting for transformation into a liquid form to be suitable for laboratory experiments. We knew that we must carefully follow each step of the resuspension process to ensure the best efficiency of the samples.

• With our gloves on and our minds focused, we prepared the equipment.as we used a trusty centrifuge, a reliable heat block, and a powerful vortexer, Additionally, we gathered pipettes, specifically a P20 and a P200, to handle the delicate genetic material.

• We repeat quantification of each sample twice using a Nanodrop instrument because this ensures that there is nothing in the water or buffer that absorbs at A260 and would artificially inflate our sample readings

Methods:

• Before opening the tube, We spun it down in a microcentrifuge for 3–5 seconds to ensure the DNA is in the bottom of the tube.
  *Note: The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.

• We added molecular grade water, to reach a final concentration of 10 ng/µL.
  *Note: Our experiments have shown that storage concentrations <1 ng/µL result in loss of material due to adherence to the plastic tube in the absence of a carrier such as tRNA.

• We vortexed it briefly.

• We incubated the tube at approximately 50°C for 15–20 min. Then we heated the tube to ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.

• We briefly vortexed the tube and centrifuged it.

• We verified the final concentration immediately by nanodrop. We used an elution buffer to blank.
  *Notes: Repeat quantification of each sample twice. When using a Nanodrop instrument, test the sample resuspension solution alone between each sample measured. This ensures that there is nothing in the water or buffer that absorbs at A260 and would artificially inflate your sample readings

PCR Amplification

• We placed a thin wall PCR tube that was placed on ice.

• The following reaction was settled:

  • 2 μL Template DNA (10 ng-500 ng)

  • 5 μl 10X Taq buffer with MgCl2

  • 1 μl dNTP mix (10 mM each nt)

  • 2.5 μL Forward Primer (10 μM stock)

  • 2.5 μL Reverse Primer (10 μM stock)

  • 0.2 μL Taq DNA Polymerase (5 units/μL)

  • 36.8 μL Sterile dH2O (variable)

• The reaction tubes were placed in a PCR machine.

• The annealing temperature was set 5°C below the primer melting temperature(TM).

• The extension step was set at 1-2 min per kilobase of product.

• The initial denaturation for 2 min at 94°C.

• Then denatured for 30 sec at 94°C.

• The primer was annealed for 30 sec at 5 Anneal primers for 30 seconds at 5°C below TM.

• The DNA was extended for 2 min at 72°C.

• The steps from 8-10 were repeated for 25-30 cycles.

• The final extension for 5 min at 72°C.

• 2 μL were tested on gel.

Digestion

Recorder: Hossam Bannis
Attendance: Youssef Amir ,Emad Hamdy , Mohamed Aboelghar
Documentation of the Day:

• Our mission Today is to prepare our plasmids for digestion using our specific restriction enzymes. Our engineered plasmids are designed to transform normal Mesenchymal Stem Cells (MSC) into our SUPER-MSCs, with their super powers.

• We are using the Digestion Protocol of NEB and to confirm correct positioning of our genetic parts, we digested each part with specific restriction enzymes. We are using six restriction enzymes due to our biobrick prefix and biobrick suffix.

We designed our genetic parts to be digested using specific restriction enzymes for each part to make sure that it will be placed in its right position in the plasmid vector. We have 3 plasmids that need to be transfected. Each one will be responsible for a specific function we provide to Mesenchymal Stem Cell (MSC) to be a SUPER-MSC. The target orientation we need to reach will be like the following tables:

Plasmid-1

Part	Length	Restriction enzymes
Syn-Notch	2740 bp	EcoRI Hind-3
Booster gene-1	2490 bp	Hind-3 Pst-1
Booster gene-2	2725 bp	Pst-1 BamHI
pcDNA 3.1- (Backbone)	5427 bp	EcoRI BamHI
Total Length	13162 bp

Table-1: Plasmid-1 consists of a pcDNA 3.1- plasmid vector ligated with 3 genetic parts : Syn-notch , Booster gene-1, and Booster gene-2. This plasmid is responsible for the expression of the Syn-Notch receptor on the surface of the transfected cell. The total length of the plasmid after ligation is 13162 bp which will be tested after ligation by gel electrophoresis.

Plasmid-2

Part	Length	Restriction enzymes
Loading System	1483 bp	EcoRI Hind-3
Exosomal Receptor	1158 bp	Hind-3 Pst-1
CX-43	2250 bp	Pst-1 BamHI
pcDNA 3.1- (Backbone)	5427 bp	EcoRI BamHI
Total Length	10079 bp

Table-2: Plasmid-2 consists of a pcDNA 3.1- plasmid vector ligated with 3 genetic parts : Loading system, Exosomal receptor, Cx43. This plasmid will be responsible for producing engineered exosomes in the transfected cell. The total length of the plasmid after ligation is 10079 bp which will be tested after ligation by gel electrophoresis.

Plasmid-3

Part	Length	Restriction enzymes
Guide RNA-BFP-switch	2725 bp	EcoRI Hind-3
New MS2	496 bp	Hind-3 Xbal
Rest of MS2 and Cas12k	2484 bp	Xbal Kpn-1
MCP-ADAR-RFP	2065 bp	Kpn-1 BamHI
pcDNA 3.1 (Backbone)	5427 bp	Eco-RI BamHI
Total Length	12845 bp

Table-3: Plasmid-3 consists of a pcDNA 3.1- plasmid vector ligated with 4 genetic parts : Guide RNA-BFP-switch, New ms2, Cas12k, and MCP-ADAR-RFP. This plasmid is responsible for producing the killing signal for the auto-reactive B-cell. The total length of the plasmid after ligation is 12845 bp which will be tested after ligation by gel electrophoresis.

Procedure:

Preparation:

• We set up the following reaction ( enzymes should be added last) :

Reagents	Volume
Nuclease free water	16u
Buffer 2:1 (10X)	2.5ul
DNA	100-500 ng
BSA	0.5ul
Restriction enzyme	0.5ul
Total	20ul




• There should be a total volume of 20ul. We mixed well and spun it down briefly.

• We incubated the restriction digest at 37 C for 30 min, and then 80 C for 20 min to heat kill the enzymes. We incubated in a thermal cycler with a heated lid

• We run a portion of the digest on a gel (8ul, 100 ng), to check that both plasmid backbone and part length are accurate.

Ligation

Recorder: Hossam Algamal
Attendance: Hossam Bannis, Ahmed wael , Khalid Hela
Documentation of the Day:

• Today our goal is to ligate our digested parts into our 3 plasmid vectors in the correct order to be ready for transfection.
  

• We did a ligation reaction to assemble our three plasmids that were ready for transfection into our Wi38 cells. We followed the ligation protocol from iGEM registry protocols and the reactions went smoothly. We are excited to see the results of the transfection and to continue the work on Wi38 cells.

• We used that restriction enzymes to digest the 3 plasmids backbone and our parts:(EcoRI - SpeI)

• We used a molar ratio of 1:3 of plasmid backbone to parts in the ligation

Ligation is the process of rearranging the digested parts in its right order in the plasmid to be ready for transfection. The genetic parts were ligated using the following protocol in order to produce 3 plasmids ready for transfection to our engineered cell.

Methods:

• We added 2ul of the digested plasmid backbone (25 ng).

• We added an equimolar amount of digested fragment (< 3 ul).

• We added 1 ul of T4 ligase buffer. Note: Do not use quick ligase.

• We added 0.5 ul of T4 ligase.

• We added 10 ul of water.

• We ligated it at 16C for 30 min. Then we heated it at 80 C for 20 min.

Colony PCR

Recorder: Mohamed Aboelghar
Attendance: Ahmed Attia, Ahmed Wael , Ahmed Shalan
Documentation of the Day:

• We prepared the following in sterile 1.5 mL microcentrifuge tube:

Component	Volume for 10 µL reaction	Master Mix volume
MilliQ water	4.6 µL	12.5 µL
OneTaq 2x Master Mix with Standard Buffer	5 µL	25 µL
Forward primer (10 µM)	0.2 µL	62.5 µL
Reverse primer (10 µM)	0.2 µL	6.25 µL




• We dispensed the 10 µL of master mix in 0.2 mL PCR tube.

• We touched a single bacterial plate with the tip of the pipette then mixed it into one of the PCR samples.

• We amplified the nucleic acid in a thermocycler according to the annealing temperature and the length of the desired amplicon.

• We visualised the results on agarose gel.

Gel electrophoresis:

Recorder: Mohamed Aboelghar
Attendance: Ahmed Attia, Ahmed Wael , Ahmed Shalan
Documentation of the Day:

• We arrived in the lab and set up the electrophoresis chamber.

• We prepared the agarose gel, EDTA buffer and EDTA.

• We poured the agarose gel into the electrophoresis chamber.

• We prepared the samples.

• We placed the samples into the wells of the agarose gel.

• After that we filled the electrophoresis chamber with a 1x TBE solution.

• We connected the electrophoresis chamber to the power supply and set the voltage to 120 volts.

• We run the gel

• We took out the gel from the electrophoresis chamber and Applied ethidium bromide to stain it.

• We visualised the fragments on the gel by a gel imager.

• After Analysing the results, we documented it.

Phase 3: Cell lab:

Wi-38 culture:

Recorder: Ahmed Attia
Attendance: Hossam Algamal ,Omar Fouad, Youssef Amir
Documentation of the Day:

Receipt of Frozen Cells and Starting Cell Cultures:

• We placed the frozen cells immediately in liquid nitrogen storage until it was ready for culture.

• When the cells were ready to culture we placed them in 37 C water until the ice crystal disappeared.

• We swapped the outside surface of the ampule with 70% ethanol, then we dispensed its content into a T75 tissue culture flask with 20mL of warm growth medium.

• We recovered the cells overnight in a 37°C, 5% CO2 humidified incubator.

• The next day, we diluted the DMSO-containing shipping/cryopreservation medium that was aspirated from the cell layer and replaced with fresh medium.

Subculture

ecorder: Ahmed Attia
Attendance: Hossam Algamal ,Omar Fouad, Youssef Amir
Documentation of the Day:

• We propagated the cells until density reached 70-80% confluence.

• Then we aspirate the medium.

• We washed the cell layer with warm 1x PBS.

• We added 15mL of Accutase then we returned the flask to the incubator for 10 -15 minutes.

• We removed the detached cells immediately to a centrifuge tube, and we rinsed the flask with warm 1X PBS to collect residual cells, and centrifuged them at 500 x g for 5 minutes (4°C).

• We resuspended the well pellet gently in a warm growth medium.

Lipofectamine transfection:

Recorder: Ahmed Gehad
Attendance:Youssef amir, Hossam Bannis , Mohamed Aboelghar
Documentation of the Day:

After arriving at the lab we proceeded to prepare the Opti MEM medium.

• After arriving at the lab we proceeded to prepare the Opti MEM medium.

• We diluted the Lipofectamine 3000 reagent in Opti-MEM medium.

• We diluted the plasmids in the Opti-MEM medium.

• We Mixed the diluted plasmids with the diluted Lipofectamine 3000 reagent.

• We Incubated the DNA-lipid complex.

• We Added the DNA-lipid complex to the cells.

• We Incubated the cells at 37°C .