iGEM 2022 — Team ualberta-canada

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}The timeline of our project.{% endblock %}
{% macro month(name) %}

## {{ name }}

{{ caller() }}

{% endmacro %}
{% macro day(date, title) %}

#### {{ date.split("-")[2] }}

**{{ title|safe }}**

{{ caller() }}

{% endmacro %}
{% block page\_content %}
{% call month("July") %}
{% call day("2022-07-9", "Antibiotic Stock, LB and LB + chloramphenicol plate preparation") %}- 34 mg/mL chloramphenicol stock solution is prepared by adding 0.378 g of 900 mg/g chloramphenicol stock in 10 mL of 95% ethanol. The stock solution is then distributed into 10 microfuge tubes as 1 mL aliquots.
- 1 L of LB is prepared for experimental use. In order to prepare 1 L of LB, 1 L of water is measured in a graduated cylinder and then is added into a flask. 25 g of solid LB broth is added to the flask along with a stir bar. The flask is placed on a magnetic stirrer to get mixed thoroughly. The mixed solution then is distributed into 5 different glass bottles as 200 mL aliquots. These bottles are then autoclaved by using liquid 45 cycle.
- 300 mL of LB is also prepared along with other solutions. 300 mL of distilled water is measured with a graduated cylinder and then is transferred into a flask. 7.5 g of solid LB broth is added to the flask along with a stir bar. The flask is placed on a magnetic stirrer to get mixed thoroughly. The solution is then autoclaved by using liquid 45 cycle.
- After the autoclaving process, the flask containing 300 mL of LB is cooled down in a 50 C incubator. Once the solution is cooled down, 300 μL of cold 34 mg/mL stock solution is added to have a final [chloramphenicol] of 34 μg/mL. The solution is mixed for a minute in a magnetic stirrer and poured into empty sterile agar plates. No flame was used in this process as the gas line of the lab was problematic. After 20 minutes, the plates were stacked into a sterile plate bag and were kept in a cold room (4C) for further use.
{% endcall %}
{% call day("2022-07-24", "Sterilization of Tubes for experimental use and additional preparation of antibiotic plates") %}- Pipette tips, 12 mL test tubes, microfuge tubes, PCR tubes, glycerol stock tubes and wooden toothpicks were autoclaved using a Gravity Filtration Cycle.
- 2 different 300 mL of LB is prepared in order to pour out kanamycin (50 μg/mL) and chloramphenicol (34 μg/mL) plates. 300 mL of distilled water is measured with a graduated cylinder and then is transferred into a flask. 7.5 g of solid LB broth is added to the flask along with a stir bar. The flask is placed on a magnetic stirrer to get mixed thoroughly. Each flask is covered by a thin foil and the solutions are then autoclaved by using the liquid 45 cycle.
- 50 mL of 100% glycerol is measured by a graduated cylinder and then is added into a 150 mL glass bottle containing 50 mL of milli-q Water to have a 50% glycerol stock solution. The solution is then autoclaved with LB solutions.
- After the autoclaving process, the flask containing 300 mL of LB is cooled down in a 50 C incubator. Once the solution is cooled down, 300 μL of cold 34 mg/mL stock solution is added to have a final [chloramphenicol] of 34 μg/mL. The solution is mixed for a minute in a magnetic stirrer and is aseptically poured into empty sterile agar plates. After 20 minutes, the plates were stacked into a sterile plate bag and were kept in a cold room (4C) for further use. The same principle is used to pour out kanamycin plates but 300 μL of kanamycin (50mg/mL) stock solution is used.
{% endcall %}
{% call day("2022-07-25", "Calibration Experiment for the 2022 iGEM Interlab Study") %}- The calibration experiment for the Interlab study was conducted based on the protocol provided by iGEM. As the wavelength values of the Plate Reader cannot be modified, the measurement is left to be done later on.
{% endcall %}
{% call day("2022-07-26", "Primer dilutions") %}- 100 mM primer dilutions are done by adding 10X volume (μL) of the nmole value of each primer. Primer stocks are vortexed vigorously both before and after the addition of corresponding volume of sterile mq water. 10 mM primer dilutions are done for the primers #35-42 by adding 10 μL of each 100 mM primer dilution into a sterile microfuge tube along with 90 μL sterile mq water. All stock solutions are kept in -20 freezer for further use
{% endcall %}
{% call day("2022-07-27", "Colony PCR to amplify HPA loci containing hpaA and hpaX along with their corresponding promoters") %}- PCR calculations are done in advance
- Two single, well-isolated E.coli BL21 colonies are picked by using a sterile tip + a P20 pipette and are added into two different microfuge tubes. These colonies are resuspended by adding 20 μL of sterile mq water
- Corresponding amount of mq water, 10X PCR Buffer, MgSO4 , 5 U/μL of Platinum Taq polymerase and 2.5 U/μL of Pfu polymerase are added respectively into a microfuge tube to create a master mix
- 3.5 μL of each primer (forward and reverse) are added into a separate microfuge tube for each PCR reaction. 61 μL of master mix then is added into each tube and mixed thoroughly by pipetting up and down
- 2 μL of each microfuge tube corresponding to resuspended tubes are transferred into 3 different tubes and are labeled as Reaction 1 Colony 1, Reaction 2 Colony 1, NC Colony 1 and Reaction 2 Colony 1, Reaction 2 Colony 2, NC Colony 2. 18.5 μL of the corresponding master mix is added into each PCR tube and mixed carefully by pipetting up and down. Corresponding settings are set-up in the Thermocycler (name the brand) by using the designated configurations : 4 minutes of initial denaturation at 96 C, a total of 30 cycles of 15 seconds of denaturation at 96 C + 30 seconds of annealing at 58 degrees (the annealing temperature is calculated by extracting 3 C from the annealing temperature of the primer with the lowest Tm value) and 60 seconds of extension step at 72 C. A final extension step is added for 5 minutes at 72C to make sure that all the products are indeed full-sized products. Reaction tubes are kept in -20 C freezer for further use.
- Also both 1 and 2% agarose gels were prepared and PCR products were run on these gels. The fragment #2 was successfully amplified as we observed a band around 500 bp yet no expected amplification occurred for the fragment #1. Please check out Joseph’s gel results.
{% endcall %}
{% call day("2022-07-28", "Test transformations for troubleshooting") %}- 1.25 uL of resuspended pJUMP27-1A plasmid [(BBa\_J428351)](http://parts.igem.org/wiki/index.php?title=Part:BBa_J428351) and one of the Interlab STudy # 2 plasmids were added into two different tubes each containing 50 uL of competent Dh5alpha cells. The protocol of transformation by chemical competence is exactly followed. (The cells were kept on ice for an hour instead of 30 minutes.) The cells were heat shocked at 47 C due to instrument limitations and this step probably caused a significant decrease in cell viability. As the lab did not contain a plate spreader, the cells were plated onto LB + CAM 34 plates using a metal spatula. These were grown overnight at 37 C. No colonies were observed the very next day.
- Heat shock was the most likely reason why the transformations failed. Yet, the amount of DNA that is added into each transformation reaction is not likely to be sufficient. From my experience, the lowest [DNA] that I used was ~ 20 ng/uL and the transformations worked perfectly fine even in that case. Yet, the maximum DNA that can be added is 3 ng which would not be sufficient for a successful transformation reaction.
{% endcall %}
{% call day("2022-07-29", "Test transformations for troubleshooting (again)") %}- This time 1, 3 and 5 uL of the resuspended plasmid (check out the exact plasmid) were added separately into 3 different microfuge tubes containing 50 uL of DH5alpha comp cells. The transformation by chemical competence protocol is exactly followed without making any changes. At the end, each pelleted and resuspended cells were plated onto LB+ CAM 34 plates and these were grown overnight at 37 C. No growth was observed the next day. Once again, the most likely reason for failed transformations is the insufficient [DNA]. There might be another possibility that the [CAM] is wrong or it is too much for successful cell growth.
- There should not be any problem with the transformation protocol. I ran around 40 different transformation reactions with this exact protocol and in every case there was at least 25 colonies. The most likely problem is the amount of DNA added is simply insufficient for a successful transformation
{% endcall %}
{% call day("2022-07-30", "Test transformations for troubleshooting (again)") %}- In this case 4 uL of resuspended plasmid [(BBa\_J23100)](http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100) is added into two microfuge tubes containing 100 uL of DH5alpha comp cells. Also, 8 uL of resuspended DNA (BBa\_I20270) is added into a different tube with 100 uL of comp cells. The transformation by chemical competence protocol is exactly followed without making any changes. The resuspended pellet of cells with 4 uL of resuspended pellet were plated onto one LB and one LB + CAM 34 plate. Cells containing 8 uL of DNA were pelleted and resuspended. 80 uL of the resuspension were plated onto two different plates (LB and LB+ CAM)
{% endcall %}
{% call day("2022-07-31", "Colony PCR to amplify HPA loci containing *hpaA* and *hpaX* along with their corresponding promoters and Pbc promoter") %}- 5-6 colonies were observed in the 13A LB+CAM 34 plate (the first successful transformation) whereas no growth was observed in 13B LB+CAM 34 plate
- There was an immense growth in each LB plate. It was a bad idea to plate everything in LB plates as well. This was done in order to see whether there was a problem with [CAM] in our plates. Eventually, all the cells, regardless of the success of transformation, grew on LB plates. What I should have done was to plate out transformants into different [CAM] to see whether a change in concentration might result in a difference.
- The PCR reaction is done based on this calculations and instructions:

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x10) |
  | Sterile mqWater | - | - | 14.93 | 149.3 |
  | PCR Buffer | 10X | 1X | 2.5 μL | 25 μL |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 μL | 25 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.4 μL | 4.0 μL |
  | Taq Pol | 5 U/μL | 0.725 U/rxn | 0.14 μL | 1.4 μL |
  | Pfu | 2.5 U/μL | 0.08 U/rxn | 0.03 μL | 0.3 μL |
  | Mix total | - | - | 20.5 | 205 |
  | Template (cell suspension) | - | - | 2.0 μL | - |
  | Final volume | - | - | 25.0 μL | - |

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Fwd primer | 10 uM | 0.5 uM | 1.25 | 4.375 |
  | Rev primer | 10 uM | 0.5 uM | 1.25 | 4.375 |

  * Add 4.375 uL of fwd and rev primers into 3 different microfuge tubes and add 69 μL master mix into those -> distribute into PCR tubes
  * 6 amplification reactions + 3 negative controls
  * Initial denaturation 4 minutes at 96
    Denaturation 15 seconds at 96
    Annealing 30 seconds at 59 for normal primers and 52.5 for Pbc primers
    Elongation 90 seconds at 72 for HPA amplification, 45 seconds for Pbc
    Final extension 5 minutes at 72
    Hold 10 degrees
  * X 30 cycles of denaturation + annealing + elongation
  * One 1% and one 1.5% of agarose gels were prepared and the products were run in these gels. The bands were not really thick, the most likely reason is that there wasn’t sufficient EtBr in the TAE buffer. Yet, it seems like we successfully amplified fragment 2 of HPA and Pbc promoter based on fragment sizes.
  * The fragment #1 of HPA still does not work. There is likely to be a problem with the primers. The ™ difference between those two is around 3-5 which should be OK. None of those primers would give primer dimer amplification as the major product. Yet, it seems like these two primers can also anneal with themselves to a certain extent. We might want to ask Dr.Stuart about what can be done in order to increase the PCR efficiency. In my opinion, we can use DMSO to increase the annealing specificity of the primers to the desired DNA region.
  * NOTE : Based on August 1’s results, it seems like the major problem is the cross-dimer of rev and primers so reducing the primer concentration might be the first thing to do.
  * I also realized that I have adding slightly more than 0.5 uM of primers
{% endcall %}
{% endcall %}
{% call month("August") %}
{% call day("2022-08-1", "Test transformations to see whether [chloramphenicol] is the problem") %}- Poulomi can provide the details
{% endcall %}
{% call day("2022-08-2", "Restreaking colonies onto a LB + Cam 34 plate + Overnight culture preparation for *E.coli* BL21") %}- Two colonies are observed in Cam 15 plate. One of the colonies is picked aseptically with a toothpick and restreaked onto a new CAM 34 plate. A significant number of colonies were observed the other day. Based on these observations, we concluded that the [antibiotic] is not the reason for low transformation efficiency.
- An overnight culture of BL21 is prepared in 5 mL of LB. The colony is picked and dumped into the tube aseptically. A negative control of LB + no colony is also prepared to see if the LB is still sterile. Only growth is observed for the LB+colony tube indicating that the LB is not contaminated. From this tube, a glycerol stock of BL21 is prepared by adding 500 uL of the overnight culture into 500 uL of sterile 50% glycerol
{% endcall %}
{% call day("2022-08-3", "PCR optimization for HPA fragment #1 and getting more PCR products of HPA #2 for purification") %}- |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x15) |
  | Sterile mqWater | - | - | 13.93 μL | 208.95 μL |
  | PCR Buffer | 10X | 1X | 2.5 μL | 37.5 μL |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 μL | 37.5 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.4 μL | 6.0 μL |
  | Taq Pol | 5 U/mL | 0.725 U/reaction | 0.14 μL | 2.1 μL |
  | Pfu | 2.5 U/mL | 0.08 U/reaction | 0.03 μL | 0.45 μL |
  | Mix total | - | - | 19.5 μL | 292.5 μL |
  | Template (cell suspension) | - | - | 3.0 μL | - |
  | Final volume | - | - | 25.0 μL | - |

  |  |  |  |  |  |  |  |  |
  | --- | --- | --- | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x2.143) | NC purposes | Extra mq water to add to 25 uL (x1) | Extra mq water to add to 25 uL (x2.143) |
  | Fwd primer | 10 μM | 0.5 μM | 1.25 | 2.68 | Add 4.02 | - | - |
  | Rev primer | 10 μM | 0.5 μM | 1.25 | 2.68 | Add 4.02 | - | - |
  | Fwd primer | 10 μM | 0.3 μM | 0.75 | 1.61 | - | 1 | 2.14 |
  | Rev primer | 10 μM | 0.3 μM | 0.75 | 1.61 | - | - |  |
  | Fwd primer | 10 μM | 0.15 μM | 0.375 | 0.8 | - | 1.75 | 3.75 |
  | Rev primer | 10 μM | 0.15 μM | 0.375 | 0.8 | - |  |  |

  ![1% Agarose Gel Electrophoresis Results of HPA Fragments #1 and #2]({{ url_for('static', filename='images/notebook/image2.webp') }})

  **1% Agarose Gel Electrophoresis Results of HPA Fragments #1 and #2.**
{% endcall %}
{% call day("2022-08-4", "Gradient PCR for HPA Fragment 1 + Final Test Transformations") %}- Gradient PCR for the HPA fragment #1 is tested in order to increase product specificity. We also added different [final] DMSO (%3 and 5%) into separate reactions to determine whether it might also increase the product efficiency.
  ![]({{ url_for('static', filename='images/notebook/image11.webp') }})
- 5% DMSO increases the product yield as it is likely to increase primer binding specificity to the template. Annealing temperature values of 61 and 58.7 C resulted in the amplification of the desired product when 3-5 %DMSO is also added. No amplification of the expected fragment is observed when the annealing temperature is dropped to 55 C. The desired fragment is obtained around 2.3 kB which is the expected product size.
{% endcall %}
{% call day("2022-08-6", "") %}- Prepared 1L of LB for experimental use
- Colony PCR for the amplification of pBC promoter from *E.coli* BL21
- Run a gradient PCR with the respective ™ values : 52.5 C and 54 C

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x6) |
  | Sterile mqWater | - | - | 14.92 μL | 89.537 μL |
  | PCR Buffer | 10X | 1X | 2.5 μL | 15 μL |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 μL | 15 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.4 μL | 2.4μL |
  | Taq Pol | 5 U/mL | 0.029 U/μL | 0.145 μL | 0.87 μL |
  | Pfu | 2.5 U/mL | 0.0032 U/μL | 0.032 μL | 0.193 μL |
  | Fwd Primer | 10 μM | 0.3 μM | 0.75 μL | 4.5 μL |
  | Rev Primer | 10 μM | 0.3 μM | 0.75 μL | 4.5 μL |
  | Mix total | - | - | 22 μL | 132 μL |
  | Template (cell suspension) | - | - | 3.0 μL | - |
  | Final volume | - | - | 25.0 μL | - |

  4 amplification reactions + 1 negative control

  Initial denaturation 4 minutes at 96
  Denaturation 15 seconds at 96
  Annealing 30 seconds at 52.5 /54 C
  Elongation 45 seconds at 72
  Final extension 5 minutes at 72
  Hold 10 C
- Denaturation + Annealing + Elongation steps are repeated for 30 cycles
- ON culture preparation of the two transformed plasmids to do minipreps. This is being done in order to observe fina [plasmid] in cells. Cultures are aseptically inoculated into separate tubes containing 5 mL of LB with a toothpick.
{% endcall %}
{% call day("2022-08-7", "") %}- Gradient PCR to amplify the GFP + B0015 terminator sequence from the iGEM plasmid [BBa\_J364001](http://parts.igem.org/Part%3ABBa_J364001) (™ values of 56 and 53 C are used for the PCR) 1 uL of DNA is added into each tube whereas 1 uL of sterile mqwater is added to the control tube.

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x4) |
  | Sterile mqWater | - | - | 16.92 μL | 67.69 μL |
  | PCR Buffer | 10X | 1X | 2.5 μL | 10 μL |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 μL | 10 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.4 μL | 1.6 μL |
  | Taq Pol | 5 U/mL | 0.029 U/μL | 0.145 μL | 0.58 μL |
  | Pfu | 2.5 U/mL | 0.0032 U/μL | 0.032 μL | 0.129 μL |
  | Fwd Primer | 10 μM | 0.3 μM | 0.75 μL | 3.0 μL |
  | Rev Primer | 10 μM | 0.3 μM | 0.75 μL | 3.0 μL |
  | Mix total | - | - | 24 μL | 96 μL |
  | Template (GFP containing plasmid) | - | - | 1.0 μL | - |
  | Final volume | - | - | 25.0 μL | - |
- 4 amplification reactions + 1 negative control
  Initial denaturation 4 minutes at 96 (could have used 2 minutes)
  Denaturation 15 seconds at 96
  Annealing 30 seconds at 53 /56 C
  Elongation 75 seconds at 72
  Final extension 5 minutes at 72
  Hold 10 C
- Denaturation + Annealing + Elongation steps are repeated for 30 cycles
- 1% gel electrophoresis is run in order to determine product sizes
  Note : Gel image is aborted as it didn’t provide corresponding details
{% endcall %}
{% call day("2022-08-8", "1% Gel electrophoresis for the PCR products of GFP") %}- ![1% Agarose Gel Electrophoresis Results of GFP + B0015 term sequence]({{ url_for('static', filename='images/notebook/image4.webp') }})

  **1% Agarose Gel Electrophoresis Results of GFP + B0015 term sequence.** Different annealing temperature values are tested in order to determine the annealing temperature specificity of the PCR reaction. The amplification reaction is conducted from the vector BBa\_J364001 provided by IGEM Kit Plate #1. A fragment size around ~ 0.9 led to the conclusion that the desired fragment was successfully amplified from the vector. DIfferent annealing temperatures did not lead to any difference in amplification.
- We determined that the corresponding GFP + term sequence is successfully amplified from the plasmid [BBa\_J364001](http://parts.igem.org/Part%3ABBa_J364001) provided by IGEM.
{% endcall %}
{% call day("2022-08-9", "Test transformations of DH5alpha with the Miniprepped IGEM plasmid DNA") %}- Two test transformations were conducted from previously isolated plasmid DNA of the successful transformation reactions in order to confirm that the [DNA] is the main reason for the failed transformation reactions and to test the efficiency of our transformation protocol. 2.5 uL of miniprepped plasmid DNA ([BBa\_J23100](http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100) and [BBa\_I20270](http://parts.igem.org/wiki/index.php?title=Part:BBa_I20270)) is added into 50 uL of DH5alpha competent cells. The transformation by chemical competence protocol was exactly followed as described in the [experiment protocols document](/experiments). The cells were pelleted and 100 uL of the recovered cells were plated onto LB+CAM34c plates. The next day, more than 100 colonies were observed in each plate. We concluded that the DNA provided by iGEM might be problematic to conduct successful transformation reactions.
{% endcall %}
{% call day("2022-08-10", "PCR Purifications of PBC, HPA fragment #1 and HPA fragment #2") %}- Accidentally, PE Buffer is added into all the tubes containing PCR products. We therefore performed another PCR reaction for PBC and HPA fragment #1. At the end, all PCR products for each different fragment were collected into a microfuge tube and PCR Purification Protocol of Qiagen is followed as described.
- Final Nanodrop Values:
  PBC = ng/μL, HPA #1= 39.9 ng/μL, HPA #2= 37.5 ng/μL
{% endcall %}
{% call day("2022-08-12", "Autoclaving More Pipette Tips and Flasks") %}- These were autoclaved in gravity filter cycle #1 (D=30 min, S=15 min)
{% endcall %}
{% call day("2022-08-13", "Overlap PCR for HPA fragments #1 and #2") %}- An alternative assembly method by overlap PCR reactions is done in order to assemble fragments HPA #1 and #2 instead of Gibson. This assembly method relies on 2 PCR reactions : the first one is being done in order to have a fully sized fragment and the second one is being done to specifically amplify this fully sized fragment (<https://www.sciencedirect.com/science/article/pii/S2215016119303358>)

  100 ng per 1 kB -> HPA fragment #1 is ~ 2.3 kB -> around 230 ng
  -> HPA fragment #2 is ~485 bp -> around 48.5 ng

  Nanodrop Values:
  HPA fragment #1 = 40 ng/μL
  HPA fragment #2 = 37.5 ng/μL

  230ng/ 40 ng/μL = 5.75 μL
  48.5 ng/37.5 ng/μL = 1.3 μL

  Rxn #1:

  |  |  |  |  |
  | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction |
  | Sterile mqWater | - | - | 31.72 μL |
  | PCR Buffer | 10X | 1X | 5 μL |
  | MgSO4 | 25 mM | 2.5 mM | 5 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.8 μL |
  | Taq Pol | 5 U/mL | 0.035U/μL | 0.35 μL |
  | Pfu | 2.5 U/mL | 0.0032 U/μL | 0.08 μL |
  | HPA fragment #1 | - | - | 5.75 μL |
  | HPA fragment #2 | - | - | 1.3 μL |
  | Final volume | - | - | 50.0 μL |

  Thermocycling conditions for the first reactions are as follows as it is described in the paper: (each amplification step is 135 seconds)
  ![]({{ url_for('static', filename='images/notebook/image14.webp') }})

  (Hilgarth and Lennigan, 2016)
- Rxn #2:

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | MM (x2.4) |
  | Sterile mqWater | - | - | 33.39 μL | 80.15 μL |
  | PCR Buffer | 10X | 1X | 5 μL | 12 μL |
  | MgSO4 | 25 mM | 2.5 mM | 5 μL | 12 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.8 μL | 1.92 μL |
  | Taq Pol | 5 U/mL | 0.025 U/μL | 0.25 μL | 0.6 μL |
  | Pfu | 2.5 U/mL | 0.0027 U/μL | 0.056 μL | 0.133 μL |
  | Fwd primer | 10 μM | 0.15 μM | 0.75 μL | 1.8 μL |
  | Rev primer | 10 μM | 0.15 μM | 0.75 μL | 1.8 μL |
  | Mix total | - | - | 46.0 μL | 110.4 μL |
  | PCR #1 product |  |  | 4.0 μL | - |
  | Final volume |  |  | 50.0 μL |  |

  Thermocycling conditions for the first reactions is altered from the paper’s reaction conditions and are as follows :

  Initial denaturation 30 seconds at 96
  Denaturation 15 seconds at 96
  Annealing 30 seconds at 58.6
  Elongation 135 seconds at 72
  Final extension 5 minutes at 72
  Hold 10 degrees

  Denaturation + Annealing + Elongation steps are repeated for 30 cycles
{% endcall %}
{% call day("2022-08-14", "Gel Electrophoresis for Overlapping PCR Reactions + More PCRs + Creating Comp Cells") %}- A single well-isolated colony is inoculated in 5 mL of LB in a test tube. This colony is grown for 7-8 hours with shaking at 37 C.
- Approximately 125 mL of LB is transferred into a sterile flask and 100 uL of the colony is added into this flask. This flask is placed into an incubator and cells are grown at 20.5 C and at 125 RPM for 15 hours. The next day, OD600 measurements of the cells were done using a spectrophotometer. Cells are grown until OD600 reaches ~ to 0.55. Upon reaching 0.55 absorbance, approximately 30 mL of cells were transferred into 4 different falcon tubes. These are kept in ice for 10 minutes. In the meantime, the centrifuge is pre-chilled to 4 C. Cells are pelleted by centrifuging at 4000 RPM for 10 minutes. Supernatants are poured out into a liquid waste and the cell pellets are resuspended with adding 10 mL of Inoue Buffer into each tube followed by pipetting thoroughly and gentle swirling. Cells are pelleted by centrifuging at 4000 RPM for 10 minutes. Once again, supernatants are removed and cells are resuspended by adding 2.5 mL Inoue Buffer into each tube followed by pipetting thoroughly and gentle swirling. Resulting resuspension is transferred into a 15mL tube and 750 uL of DMSO is added into the solution. The tube is incubated on ice for 10 minutes and the solution is distributed into 750 uL aliquots and these microfuge tubes are kept in -80 freezer

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x5) |
  | Sterile mqWater | - | - | 16.72 μL | 83.6 μL |
  | PCR Buffer | 10X | 1X | 2.5 μL | 12.5 μL |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 μL | 12.5 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.4 μL | 2 μL |
  | Taq Pol | 5 U/mL | 0.029 U/μL | 0.145 μL | 0.725 μL |
  | Pfu | 2.5 U/mL | 0.0032 U/μL | 0.032 μL | 0.161 μL |
  | Fwd Primer | 10 μM | 0.3 μM | 0.75 μL | 3.75 μL |
  | Rev Primer | 10 μM | 0.3 μM | 0.75 μL | 3.75 μL |
  | Mix total | - | - | 23.8 μL | 119 μL |
  | Template (GFP containing plasmid) | - | - | 1.2 μL | - |
  | Final volume | - | - | 25.0 μL | - |
{% endcall %}
{% call day("2022-08-15", "GFP PCRs and Test Transformations for the Comp Cells") %}- ![1% Agarose Gel Electrophoresis Results of GFP + B0015 term sequence along with the overlap PCR reaction to assemble 2 HPA fragments]({{ url_for('static', filename='images/notebook/image5.webp') }})

  **1% Agarose Gel Electrophoresis Results of GFP + B0015 term sequence along with the overlap PCR reaction to assemble 2 HPA fragments.** The amplification reaction for GFP is conducted from the vector [BBa\_J364001](http://parts.igem.org/Part%3ABBa_J364001) provided by IGEM Kit Plate #1. Very faint bands for GFP amplicons are observed. Overlapping PCR reaction to assemble two HPA fragments did not result in the expected amplification product as the major product seemed to be the same size as the HPA fragment #2.
{% endcall %}
{% call day("2022-08-16", "PCR Amplification of GFP") %}- ![1% Agarose Gel Electrophoresis Results of GFP + B0015 term sequence]({{ url_for('static', filename='images/notebook/image3.webp') }})

  **1% Agarose Gel Electrophoresis Results of GFP + B0015 term sequence.** The amplification reaction for GFP is conducted from the vector [BBa\_J364001](http://parts.igem.org/Part%3ABBa_J364001) provided by IGEM Kit Plate #1. The bands at 900 bp indicate that GFP is successfully amplified with a high [amplicon]
{% endcall %}
{% call day("2022-08-19", "PCR Amplification of individual Pbc fragments for the assembly with bam genes + Gibson for individual HPA fragments") %}- PCR reactions were inconclusive due to lack of Taq polymerase stock
{% endcall %}
{% call day("2022-08-20", "PCR amplification of the HPA Gibson fragment") %}- PCR amplification of the full sized HPA fragment from the Gibson reaction is performed. The Gibson reaction is diluted 1:4 with mq water before the PCR

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x5) |
  | Sterile mqWater | - | - | 29.77 μL | 148.86 μL |
  | PCR Buffer | 10X | 1X | 5 μL | 25 μL |
  | MgSO4 | 25 mM | 2.5 mM | 5 μL | 25 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.8 μL | 4 μL |
  | Taq Pol | 5 U/mL | 0.035 U/μL | 0.35 μL | 1.75 μL |
  | Pfu | 2.5 U/mL | 0.0039 U/μL | 0.078 μL | 0.389 μL |
  | Fwd Primer | 10 μM | 0.4 μM | 2 μL | 10 μL |
  | Rev Primer | 10 μM | 0.4 μM | 2 μL | 10 μL |
  | Mix total | - | - | 45 μL | 225 μL |
  | Template (Gibson resuspension) | - | - | 5 μL | - |

  2 ul Gibson product : 8 ul mq water

  Three test tubes for the Gibson PCR :
  #1 : 5 uL diluted Gibson product
  #2 : 3.5 uL diluted Gibson product + 1.5 uL mqwater
  #3 : 1.5 uL diluted Gibson product + 3.5 uL mqwater
  #4 : 4 ul overlapping PCR product + 1 ul mqwater
  #5 : 5 uL of mqwater is used for the negative control.

  Primers : Golden Gate 1 of HPA +Golden Gate 2 of HPA

  TMs : 66.7 for fwd , 68.3 for rev -> use 64 C

  Initial denat : 2 min at 96

  Gel Results :
  ![1% Agarose Gel Electrophoresis Results of Gibson Reaction Products used to assemble individual HPA fragments]({{ url_for('static', filename='images/notebook/image10.webp') }})

  **1% Agarose Gel Electrophoresis Results of Gibson Reaction Products used to assemble individual HPA fragments.** None of the amplification of the Gibson products did not result in the expected product size of (2.8 kB). HPA fragments # 1 and # are used to confirm that product size of Gibson reaction is different from those fragments

  Based on the gel image, we confirmed that the Gibson reaction did not go as planned and no assembly of the two individual fragments to give a full-sized HPA fragment has occurred since we could have had a full-length fragment at the end of the PCR if the Gibson reaction was successful.

  Going forward, I decided to amplify the full-length HPA fragment right from the *E.coli* BL21 genome. This region does contain an internal PaqCI cut site; yet, once it is digested, the ends should only be compatible with one another and not with any other digested fragment. Based on that, our cloning strategy should remain the same although we get the fragment with the internal digestion site.
- |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x 2.4) |
  | Sterile mqWater | - | - | 29.77 μL | 71.45 μL |
  | PCR Buffer | 10X | 1X | 5 μL | 12 μL |
  | MgSO4 | 25 mM | 2.5 mM | 5 μL | 12 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.8 μL | 1.92 μL |
  | Taq Pol | 5 U/mL | 0.035 U/μL | 0.35 μL | 0.84 μL |
  | Pfu | 2.5 U/mL | 0.0039 U/μL | 0.078 μL | 0.187 μL |
  | Fwd Primer | 10 μM | 0.4 μM | 2 μL | 4.8 μL |
  | Rev Primer | 10 μM | 0.4 μM | 2 μL | 4.8 μL |
  | Mix total | - | - | 45 μL | 108 μL |
  | Template (cell resuspension) | - | - | 5 μL | - |

  Two reaction tubes + NC tube with the leftover

  Gel image :
  ![1% Agarose Gel Electrophoresis Results of the full-sized HPA fragment]({{ url_for('static', filename='images/notebook/image8.webp') }})

  **1% Agarose Gel Electrophoresis Results of the full-sized HPA fragment.** A colony PCR reaction is conducted to amplify the full-length HPA fragment from E.coli BL21. The resulting product gave a size around 2.8 kB which is the expected length for the full-length HPA fragment.

  Based on the gel picture, we can confirm that we got the full-sized HPA fragment. Individual HPA fragments can also be observed on the gel which are in different sizes than the full-length fragment. This fragment can be purified and be used for cloning.
{% endcall %}
{% call day("2022-08-26", "PCR amplification of pJUMP27-1A plasmid to introduce BsaI cut sites and PCR Purification of some products") %}- The low-copy plasmid pJUMP27-1A provided by IGEM is PCR amplified with the tailed-primers containing each BsaI cut sites. The ends of the product should be compatible with the full-sized HPA fragment once digested by BsaI.

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x 2) |
  | Sterile mqWater | - | - | 32.27 μL | 65.54 μL |
  | PCR Buffer | 10X | 1X | 5 μL | 10 μL |
  | MgSO4 | 25 mM | 2.5 mM | 5 μL | 10 μL |
  | dNTPs | 25 mM | 0.4 mM | 0.8 μL | 1.6 μL |
  | Taq Pol | 5 U/mL | 0.035 U/μL | 0.35 μL | 0.7 μL |
  | Pfu | 2.5 U/mL | 0.0039 U/μL | 0.078 μL | 0.156 μL |
  | Fwd Primer | 10 μM | 0.4 μM | 2 μL | 4 μL |
  | Rev Primer | 10 μM | 0.4 μM | 2 μL | 4 μL |
  | Mix total | - | - | 45 μL | 108 μL |
  | Template (resuspended plasmid) | - | - | 2.5 μL | - |

  One reaction tube + one NC

  Reaction conditions :

  Initial denaturation 120 seconds at 96
  Denaturation 15 seconds at 96
  Annealing 30 seconds at 52.0
  Elongation 170 seconds at 72
  Final extension 5 minutes at 72
  Hold 10 degrees

  Gel Results :
  ![1% Agarose Gel Electrophoresis Results of pJUMP-271A with BsaI ends]({{ url_for('static', filename='images/notebook/image7.webp') }})

  **1% Agarose Gel Electrophoresis Results of pJUMP-271A with BsaI ends.** pJUMP-271A plasmid provided by IGEM kit Plate #1 is amplified using two primers with BsaI cut sites as tails. The linearized product gave a size around 3 - 3.1 kB which is the expected size of the plasmid

  As it can be seen from the gel, the product is around ~ 3- 3.1 kB which is the expected size for the amplicon.

  Going forward, the purification of full-sized HPA, GFP and pJUMP27-1A w/ BsaI ends is performed based on the protocol provided by Qiagen PCR Purification Kit.

  The products of GFP and pJUMP27-1A are eluted with 40 uL of EB to have more concentrated DNA whereas 50 uL of EB is used to get the HPA fragment.

  Final NanoDrop concentrations :

  HPA = 100 ng/uL
  GFP =
  pJUMP27-1A w/ Bsa = 40 ng/uL
{% endcall %}
{% call day("2022-08-28", "PCR amplification of individual pBC fragments") %}- E.coli BL21 is PCR amplified with each corresponding primer containing either Golden Gate or Gibson Tails. (Gibson Tails are based on the bam genes)

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x 2) |
  | Sterile mqWater | - | - | 43.16 μL | 258.95 μL |
  | PCR Buffer | 10X | 1X | 7.5 μL | 45 μL |
  | MgSO4 | 25 mM | 2.5 mM | 7.5 μL | 45 μL |
  | dNTPs | 25 mM | 0.4 mM | 1.2 μL | 7.2 μL |
  | Taq Pol | 5 U/mL | 0.035 U/μL | 0.525 μL | 3.15 μL |
  | Pfu | 2.5 U/mL | 0.0039 U/μL | 0.117 μL | 0.7 μL |
  | Mix total | - | - | 60 μL | 108 μL |
  | Fwd Primer | 10 μM | 0.7 μM | 5 μL | - |
  | Rev Primer | 10 μM | 0.7 μM | 5 μL | - |
  | Template (resuspended plasmid) | - | - | μL | - |

  Reaction conditions:

  Initial denaturation 240 seconds at 96
  Denaturation 15 seconds at 96
  Annealing 30 seconds at 56.0
  Elongation 45 seconds at 72
  Final extension 5 minutes at 72
  Hold 10 degrees
  ![]({{ url_for('static', filename='images/notebook/image6.webp') }})
{% endcall %}
{% call day("2022-08-29", "GG Assembly to ligate HPA locus into the target vector pJUMP27-1A with BsaI ends") %}- For Golden Gate

  Full-sized HPA fragment : 2754 bp
  Plasmid ~ 3079 bp

  80 ng of plasmid -> 40 ng/ul -> 2 ul of plasmid
  Molar ratio insert;plasmid -> 80x 2754/3079 = 71.6 ng of insert
  2:1 ratio of insert:plasmid -> 71.6 ng x 2 = 143.2 ng
  HPA insert -> 100 ng/uL
  143.2/100 = 1.43 uL
  1 uL GG assembly mix
  2 uL T4 Buffer
  20 - all = 13.47 uL mq water

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | Reaction | Negative Control |
  | Mq Water | - | - | 13.57 uL | 15 uL |
  | Vector | - | - | 2 uL | 2 uL |
  | Insert (HPA) | - | - | 1.43 uL | - |
  | T4 Ligase Buffer | 10x | 1x | 2 uL | 2 uL |
  | BsaI HF Solution | - | - | 1 uL | 1 uL |
  | Total | - | - | 20 uL | 20 uL |

  3 uL of the reaction product is used for transforming 75 uL of DH5alpha comp cells
- We also transformed 100 uL of Dh5alpha comp cells with IGEM plasmid [BBa\_J428358](http://parts.igem.org/Part%3ABBa_J428358)
- Transformation for chemical competence procedure is followed
{% endcall %}
{% call day("2022-08-30", "GG Assembly to ligate HPA locus into the target vector pJUMP27-1A with BsaI ends") %}- No colonies for the Golden Gate reaction yet the transformation of the IGEM plasmid worked. Either Golden Gate reaction failed or there is something wrong with the kanamycin plates (the stock might be different or kan50 is a lot for a low copy plasmid)
- Running the components on an agarose gel :
  ![1% Agarose Gel Electrophoresis Results of the Golden Gate Product]({{ url_for('static', filename='images/notebook/image15.webp') }})

  **1% Agarose Gel Electrophoresis Results of the Golden Gate Product.** As the transformation reactions failed with the corresponding golden gate reaction product, it was run on a gel to compare it with linearized plasmid and HPA fragment. The gel indicates 3 different conformations of a plasmid structure : OC,CCC,SC

  It might be possible that the bands corresponding to the Golden Gate Product might be OC, CCC, SC conformations of the plasmid. That would indicate a successful Golden Gate reaction as the linear fragments are circularized

  On top agar antibiotic plating is used ->
  For kan25 (25ug/mL) = Mix 4 uL of Kan 50 mg/mL stock with 196 uL of LB -> Plate 75 uL into each side of the plate both for plating the supernatant and the pellet
{% endcall %}
{% call day("2022-08-31", "GG Assembly to ligate HPA locus into the target vector pJUMP27-1A with BsaI ends") %}- Successful colonies observed in both kan25 and kan12.5 plates, the next step is colony PCR to screen the insert
{% endcall %}
{% endcall %}
{% call month("September") %}
{% call day("2022-09-5", "Colony PCR for screening and patching out the colonies onto new plates") %}- 3 single well isolated colonies from each kan25 and kan12.5 plates were resuspended in 20 uL of mqwater.

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x 7.2) |
  | Sterile mqWater | - | - | 15.40444444 | 110.912 |
  | PCR Buffer | 10X | 1X | 2.5 | 18 |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 | 18 |
  | dNTPs | 25 mM | 0.4 mM | 0.4 | 2.88 |
  | Taq Pol | 5 U/mL | 0.032 U/μL | 0.16 | 1.152 |
  | Fwd Primer | 10 μM | 0.3 μM | 0.75 | 6.48 |
  | Rev Primer | 10 μM | 0.3 μM | 0.75 | 6.48 |
  | Mix total | - | - | 22.5 μL | 162μL |
  | Template (resuspended plasmid) | - | - | 2.5 μL | - |

  2.5 uL of mq water is used for the NC

  Initial denaturation 240 seconds at 96
  Denaturation 15 seconds at 96
  Annealing 30 seconds at 63.5
  Elongation 150 seconds at 72
  Final extension 5 minutes at 72

  On top agar antibiotic plating is used to have the following plates : Kan100 ug/ml, Kan 50ug/ml, Kan 25ug/ml. LB + Kan 50mg/mL combinations were prepared respectively for the corresponding plates : 138 uL LB + 12 uL Kan 50mg/mL stock, 144 uL LB + 6 uL Kan 50mg/mL stock, 147 uL LB + 3 uL Kan 50mg/mL stock

  These antibiotic mixtures were then spread onto 3 different LB plates using a glass cell spreader.

  Sterile toothpicks were dipped into each resuspension and those were patched out onto each plate

  ![1% Agarose Gel Electrophoresis Results of the Colony PCR to screen for the HPA insert]({{ url_for('static', filename='images/notebook/image9.webp') }})

  **1% Agarose Gel Electrophoresis Results of the Colony PCR to screen for the HPA insert.** Successful transformant colonies were diluted in 20 uL of mqwater and a colony PCR is ran to confirm for the presence of HPA insert inside the plasmid.

  NOTE : Colonies # 3 and 6 contain the corresponding HPA insert which is around 2.8 kB. We decided to use strain corresponding to colony #3 as it resulted in a thicker band for the insert.
- |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x 14.4) |
  | Sterile mqWater | - | - | 14.90444444 | 214.624 |
  | PCR Buffer | 10X | 1X | 2.5 | 36 |
  | MgSO4 | 25 mM | 2.5 mM | 2.5 | 36 |
  | dNTPs | 25 mM | 0.4 mM | 0.4 | 5.76 |
  | Taq Pol | 5 U/mL | 0.032 U/μL | 0.16 | 2.304 |
  | Pfu | 2.5 U/mL | 0.0036 U/μL | 0.03555555556 | 0.512 |
  | Mix total | - | - | 20.5 μL | 295.2 μL |
  | Fwd Primer | 10 μM | 0.4 μM | 1 | 2.4 |
  | Rev Primer | 10 μM | 0.4 μM | 1 | 2.4 |
  | Mix total | - | - | 22.5 μL | - |
  | Template (resuspended plasmid) | - | - | 2.5 μL | - |

  ![]({{ url_for('static', filename='images/notebook/image12.webp') }})

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x3) |
  | Sterile mqWater | - | - | 17.7 | 53.1 |
  | PCR Buffer | 10X | 1X | 2.5 | 7.5 |
  | MgSO4 | 50 mM | 2.5 mM | 1.25 | 3.75 |
  | dNTPs | 25 mM | 0.4 mM | 0.4 | 1.2 |
  | Taq Pol | 5 U/mL | 0.033 U/μL | 0.16 | 0.5 |
  | Fwd Primer | 10 μM | 0.4 μM | 1 | 3 |
  | Rev Primer | 10 μM | 0.4 μM | 1 | 3 |
  | Mix total | - | - | 24 μL | 72 |
  | Template (resuspended plasmid) | - | - | 1 | - |

  ![]({{ url_for('static', filename='images/notebook/image13.webp') }})

  |  |  |  |  |  |
  | --- | --- | --- | --- | --- |
  | Component | [Stock] | [Final] | 1 Reaction | Master Mix (x 13) |
  | Sterile mqWater | - | - | 17.64833333 | 229.4283333 |
  | PCR Buffer | 10X | 1X | 2.5 | 32.5 |
  | MgSO4 | 25 mM | 2.5 mM | 1.25 | 16.25 |
  | dNTPs | 25 mM | 0.4 mM | 0.4 | 5.2 |
  | Taq Pol | 5 U/mL | 0.032 U/μL | 0.165 | 2.145 |
  | Pfu | 2.5 U/mL | 0.0036 U/μL | 0.03666666667 | 0.4766666667 |
  | Mix total | - | - | 22 μL | 295.2 μL |
  | Fwd Primer | 10 μM | 0.4 μM | 1 | 3.25 |
  | Rev Primer | 10 μM | 0.4 μM | 1 | 3.25 |
  | Mix total | - | - | 24 μL | - |
  | Template (resuspended plasmid) | - | - | 1 μL | - |
{% endcall %}
{% endcall %}
{% endblock %}


{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}The protocols for the experiments that we used.{% endblock %}
{% macro technique(name) %}

## {{ name|safe }}

---

{{ caller() }}

{% endmacro %}
{% block page\_content %}
{% call(section) technique("Preparing LB + corresponding Plates") %}

1. Measure corresponding amount of distilled water (usually 1 L when preparing LB for experimental use and 500 mL for preparing plates) into a graduated cylinder and transfer it into an erlenmeyer flask
2. Add a stir bar into the flask and place it onto a magnetic stirrer
3. As we have access to LB broth, just weigh out 2.5% of this broth and add into the flask. If no access to this compound, weigh out 1% (w/v) of tryptone and NaCl and 0.5% (w/v) of yeast extract separately and add them into the beaker/flask (For 1 L of LB, that makes 10 g of tryptone, 10 g of NaCl and 5 g of yeast extract)
4. Wait until the solution is completely dissolved
5. If preparing LB for agar plates, add 1.5% (w/v) of bacto agar into the flask and swirl it to break up clumps of agar
6. Cap the flask with a foil and attach a piece of autoclave tape on top of the foil
7. If preparing LB for experimental use, distribute the solution into 150/250 mL graduated glass bottles (be careful not to add more than 125/200 mL into each as the solution can bump out while being autoclaved)
8. Put caps into each bottle but do not tighten completely and add autoclave tapes onto them
9. Autoclave the liquids accordingly (only people with proper autoclave training must use it)
10. LB media for experimental purposes is ready to use as soon as cooled down
11. After autoclaving cycle, transfer the flask containing bacto agar into a 50 °C water bath.
12. Wait until the solution cools down (usually between 20 - 40 minutes)
13. While waiting, prepare and label corresponding sterile petri plates
14. Add corresponding antibiotics into the solution by using a micropipette once cooled down (usually 0.1% (v/v) of antibiotic will be added)
15. Put the flask onto a magnetic stirrer and stir around 1-2 minutes to mix antibiotics with the solution
16. Aseptically pour down the solution into petri plates (be careful but as fast as possible as the agar will solidify once cooled down)
17. If necessary, get rid of any bubbles forming by gently touching the bubble with the flame

{% endcall %}
{% call(section) technique("Transformation by Chemical Competence (Transforming an IGEM Plasmid)") %}

1. Pipette 5 µL of dH₂O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye.
2. Transform all 5 µL of the resuspended DNA into 100 µL of competent *E. coli* DH5⍺
3. Incubate the cells on ice for 30 minutes after DNA is added
4. Heat shock at 42.5 °C for 1 minute
5. Add 1 ml LB to tube and let cells recover at 37°C with shaking for an hour
6. Pellet the cells at 14000 RPM for a minute (this is especially crucial when transforming an iGEM Plasmid as the DNA provided is not a lot), discard the supernatant but leave around 100 µL of the supernatant, resuspend the pellet with the leftover supernatant and plate 100 µL of the resuspension onto the corresponding plate
7. Grow ON at 37 °C
8. The next day pick up the plates from the incubator and store them in the -4 °C fridge

{% endcall %}
{% call(section) technique("Transformation by Chemical Competence (Transforming a Miniprepped Plasmid/Gibson - GG Product)") %}

1. Transform 2 µL of the resuspended DNA into 50 µL competent *E. coli* strain
2. Incubate the cells on ice for 30 minutes with DNA added
3. Heat shock at 42.5 °C for 1 minute
4. Add 1 ml LB to tube and let cells recover at 37 °C with shaking for an hour
5. Plate cells on LB + corresponding antibiotics and grow overnight at 37 °C incubator (volume to be plated depends on transformation efficiency)
6. Another thing to do might be pelleting the cells at max speed for a minute after plating 100 µL of transformed cells, aseptically resuspending the pellet in 100 µL of LB and plating another 100 µL onto another plate)
7. Grow ON at 37 °C
8. The next day, pick up the plates from the incubator and store them in the -4 °C fridge

{% endcall %}
{% call(section) technique("Minipreps to Collect plasmid DNA from the cells (based on Qiagen Miniprep Kit)") %}

1. Pick a single well-isolated colony aseptically and resuspend in 5 mL of LB + corresponding antibiotics and grow overnight (for ~16 hours) at 37 ˚C with shaking
2. The next day, transfer 1.5 mL of the solution into a microfuge tube and pellet the cells by centrifuging at max speed for a minute
3. Get rid of the supernatant and transfer another 1.5 mL of the overnight culture into the microfuge tube
4. Centrifuge for another 30 seconds and remove the supernatant with a pipette
5. Resuspend the pellet in 250 µL of Buffer P1 (with RNase already added) (mix well by vortexing or pipetting up and down)
6. Add 250 µL of Buffer P2 and mix by gentle inversion 4-6 times (by no means should you vortex or pipette up and down this solution) and let it sit for 2-3 minutes
7. Add 350 µL of Buffer N3 + gentle inversion 4-6 times
8. Centrifuge for 10 minutes (~13500 RPM)
9. Label a QIAprep spin column and put it into a 2 mL collection tube
10. Transfer the supernatant into the spin column (usually around 700 - 800 µL)
11. Centrifuge at maximum speed for a minute + discard the flow-through + place it back
12. Add 750 µL of Buffer PE + maximum speed centrifuge + discard the flow-through
13. Centrifuge for another 1 - 2 minutes to get rid off any remaining material
14. Transfer the spin column into a labeled microfuge tube
15. Add either 50 µL of EB solution or dH₂O into the center of the membrane (use the one that works with higher efficiency) + let it sit for 2 - 3 minutes
16. If the plasmid to be isolated is large, preheat the solution to be added to 50 ˚C and add the heated solution into the center
17. Centrifuge at max speed for 1 minute + discard the spin column and quantify the DNA by using a NanoDrop
18. Blank the NanoDrop with 2 µL of Milli-Q water
19. Load 2 µL of your sample and note the absorption values
20. Collected plasmid DNA can be stored in eppendorf tubes at -20 ˚C

{% endcall %}
{% call(section) technique("Preparing glycerol stocks of any *E. coli* strain") %}

1. Pick a single well-isolated colony aseptically and resuspend in 5 mL of LB + corresponding antibiotics and grow overnight (for ~16 hours) at 37˚C with shaking (200-250 RPM)
2. The next day, aseptically add 500 µL of the culture into a glycerol stock tube
3. Add 500 µL of 50% glycerol into the tube, mix thoroughly by pipetting up and down and store it at -80 ˚C

{% endcall %}
{% call(section) technique("Preparing an ON culture of any *E. coli* strain") %}

1. Wipe down the bench with 70% ethanol
2. Get a sterile test tube and turn on the Bunsen Burner
3. Add 5 mL of LB media in the tube aseptically using a plastic pipette
4. Add the appropriate volume of antibiotics if necessary (NOTE: Use chloramphenicol at a final concentration of 34 µg/mL and kanamycin at 25-50 µg/mL)
5. Pick a single well-isolated colony aseptically using a toothpick and inoculate into the liquid media
6. Grow overnight (for ~16 hours) at 37 ˚C with shaking (200-250 RPM)

{% endcall %}
{% call(section) technique("PCR and Agarose Gel electrophoresis") %}

1. Resuspend an isolated colony into 20 μL of Milli-Q water
2. Calculate in advance volumes of all the components required for the PCR reaction
3. Prepare your master mix by keeping the microfuge tube on ice (add water first and DNA polymerase the last)
4. If you're using different primers for your PCRs, you can simply create one Master Mix and then distribute the corresponding amount into different microfuge tubes containing each primer pair set
5. Add the appropriate volume of Master Mix into PCR tubes and add 3.0 μL of the resuspended DNA if using a colony PCR - If you're adding purified DNA instead of a colony PCR please do the calculations and add the appropriate amount of DNA (keep PCR tubes on ice as well)
6. Make sure to include a NC everytime with no DNA and with the appropriate amount of sterile Milli-Q water
7. Setup the thermocycler based on the details of your PCR reaction and the Tm of your primers

{% endcall %}
{% call(section) technique("Setting up an agarose gel") %}

1. To make 1% agarose gel, measure 0.25 mg agarose in 25 mL of 1X TAE buffer into a flask
2. Dissolve the agarose into the buffer by heating it up in a microwave (pay attention to the solution as it might bump out once it starts boiling)
3. Cool down the solution by putting the edges of the flask under the water
4. Quickly but carefully add 0.5 µg/mL (1.5 µL in this case) of EtBr into the solution in the fume hood and swirl to mix the solution
5. Pour down the agarose into the gel tray and put the corresponding comp onto it
6. Let it sit for 15 minutes for the gel to be solidified
7. Run your samples (mix the dye and DNA on a parafilm)
8. Run the gel for ~15-20 minutes at 100 Volts and for ~35-40 minutes at 50 Volts (until the yellow band on the ladder disappears)
9. Get the gel and visualize it in a computer

{% endcall %}
{% call(section) technique("Primer Resuspension & Dilution") %}

1. Calculate the amount of Milli-Q water required to create a 100X dilution
2. Vortex primer stock tubes for 5-10 seconds vigorously
3. Add the corresponding amount of Milli-Q water and pipette up and down to mix thoroughly
4. Get some sterile microfuge tubes, label them accordingly, add 10 µL of 100X primer resuspension and 90 µL of sterile Milli-Q water, repeat this process for each primer. These will give us stocks with 10X working concentration
5. Keep all primer resuspensions + dilutions in an ice box at -20 ºC

{% endcall %}
{% call(section) technique("Creating Competent Cells (Modified Inoue Method)") %}

1. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37 ºC. Transfer the colony into 25 mL of LB broth in a 250 mL flask. Incubate the culture for 6-8 hours at 37 ºC with vigorous shaking (250-300 rpm).
2. At about 6 o'clock in the evening, use this starter culture to inoculate a subculture: the flask receives 50 µL of the incubated culture and 125 µL of LB. Incubate it overnight at 18-22 ºC with moderate shaking.
3. The following morning, read the OD600 and continue to monitor the OD every 25-30 minutes until it reaches to 0.55
4. Distribute the culture into 4 different falcon tubes ~30 mL into each. Transfer those tubes to an ice-water bath for 10 minutes.
5. Harvest the cells by centrifugation at 4000 RPM for 10 minutes at 4 ºC. (cool down the centrifuge to 4 ºC prior to this step)
6. Pour off the supernatant, resuspend cells by gently pipetting up down in each tube with 10 mL of ice-cold Inoue transformation buffer.
7. Harvest the cells by centrifugation 4000 RPM for 10 minutes at 4 ºC.
8. Pour off the supernatant, resuspend the cells in each tube gently in 2.5 mL of ice-cold Inoue transformation buffer
9. Combine the resuspensions into one falcon tube ~10 mL
10. Add 0.7 - 0.75 mL of DMSO drop-wise while swirling the culture, ~7% final volume. Mix the bacterial suspension by swirling and then store it in ice for 10 minutes
11. Working quickly, dispense aliquots of the suspensions into chilled, sterile microfuge tubes - aliquots of 250 - 500 uL
12. Keep those tubes at -80 ºC for further use

NOTE: Volumes are subject to change based on our decision of how much comp cell to create; yet, keep in mind that each volume to be added will also change in this case
{% endcall %}
{% call(section) technique("Kirby-Bauer Media Preparation") %}

1. Add 38 g of Meuller Hinton Agar in 1 liter of distilled water
2. Add 20% Glucose
3. Add 33 µl of Methylene blue
4. Using heated magnetic stir plate - stir contents until dissolved and solution is boiling
5. Immediately remove once boiling
6. Autoclave contents on liquid 45 setting (~1h, 15min)
7. Pour out plates under sterile conditions. Let plates cool 30-40 minutes before use. If not in use, place plates in bag then leave at 4 ºC

{% endcall %}
{% call(section) technique("Kirby-Bauer Fungi Resuspension & Plating") %}

1. Re-streak single colony isolate onto new plate - incubate overnight. Must be streaked, not spread to ensure SCI is created
2. Take 1 colony from sub-culture plate and inoculate it in 0.9% saline (~2ml). Note: 0.9% saline can be made with dH₂O + NaCl -> Autoclave L45 Setting
3. Vortex at low speed for 5 seconds
4. Measure optical density at 530 nm. Must be between 0.09-0.12. If higher, dilute by adding more saline. If lower, concentrate by adding more colonies (this is not optimal)
5. Soak a sterile swab in inoculated saline (~20 sec)
6. Bring swab to top of tube, using moderate pressure, press the cotton into the side of the tube, rotating the swab around the whole top of the tube (helps prevent excess moisture from going onto the plate)
7. Using the same swab, streak out the colonies. When streaking, ensure the swab reaches the ends of the plate, and no gaps appear between new swab lines. Swab the whole plate in a downwards direction, then from left to right, then diagonally.
8. Add Soaked paper discs in 4 regions based on their concentration of Nisin
9. Incubate overnight
10. Take a picture of inhibition zones and draw them on top of the plate at 12 hours, 24 hours, 36 hours, and 48 hours. (24 hour mark is crucial, others are for more data, but not absolutely necessary)

{% endcall %}
{% call(section) technique("Kirby-Bauer Disk Preparation") %}

1. Either obtain paper disks from a professor, or make your own using paper and a hole puncher
2. In a small tin-foil covered beaker, autoclave the paper disks
3. Tape down the tin-foil using autoclave tape
4. Ensure forceps are sterile - dip in ethanol, then light on fire to remove ethanol - let cool before touching disk
5. Using forceps, soak 4 paper disks in the 4 different Nisin concentrations and place on the plate in 4 different quadrants. Ensure disks are placed evenly spaced from one another, and each has a reasonable amount of room for the zone of inhibition to form around it on all sides.

{% endcall %}
{% call(section) technique("Kirby-Bauer Test") %}

| Quadrant 1 - Stock nisin | Quadrant 2 - 1/10 nisin | Quadrant 3 - 1/20 nisin | Quadrant 4 - negative control |
| --- | --- | --- | --- |
| 100 μL nisin | 10 μL nisin + 90 μL ddH₂O | 5 μL nisin + 95 μL ddH₂O | 100 μL ddH₂O |

1. Streak fungi on plates as described above
2. Immediately place soaked paper disks in their 4 quadrants as described above
3. Incubate at 37 ℃ overnight (~24 hours) as described above. Additional measurements as described above are optional and to be done only if time permits
4. When reviewing zones of inhibition, draw the zone on the top of the plate in sharpie
5. Using a ruler, measure the diameter of the zone
6. Determine whether the fungi is resistant
7. Take note of any growth within the zone. No need to culture these further, but could be useful information for future analysis

{% endcall %}
{% endblock %}