iGEM 2019 — Team UM_Macau

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# Team:UM Macau/Notebook

[UM\_Macau](https://2019.igem.org/Team%3AUM_Macau)

* Team

  [Team Members](https://2019.igem.org/Team%3AUM_Macau/Team)
  [Attribution](https://2019.igem.org/Team%3AUM_Macau/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AUM_Macau/Description)
  [Experiment Design](https://2019.igem.org/Team%3AUM_Macau/Experiments)
  [Results](https://2019.igem.org/Team%3AUM_Macau/Results)
  [Notebook](https://2019.igem.org/Team%3AUM_Macau/Notebook)
  [Modeling](https://2019.igem.org/Team%3AUM_Macau/Model)
  [Demonstrate](https://2019.igem.org/Team%3AUM_Macau/Demonstrate)
* Parts

  [Basic Part](https://2019.igem.org/Team%3AUM_Macau/Basic_Part)
  [Composite Part](https://2019.igem.org/Team%3AUM_Macau/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AUM_Macau/Part_Collection)
* [Lab Safety](https://2019.igem.org/Team%3AUM_Macau/Safety)
* Human Practice

  [Integrated Human Practice](https://2019.igem.org/Team%3AUM_Macau/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3AUM_Macau/Public_Engagement)
  [Collaboration](https://2019.igem.org/Team%3AUM_Macau/Collaborations)
* Achievement

  [Judging Form](https://igem.org/2019_Judging_Form?team=UM_Macau)
  [Medal Criteria](https://2019.igem.org/Template%3AUM_Macau/Medal_Criteria)

# Notebook

[![](https://static.igem.org/mediawiki/2019/3/36/T--UM_Macau--homepicdownarrow.png)](#pagestart)

Lablogs
Protocols

May
June
July
August
September
October

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May 20, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | gBlocks PCR amplification: IDT-OMPHT,NSP4-tyrosinase,Mefp-5 |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | PCR reaction: Prepare reaction mix on ice. Primers for IDT-OMPHT are 100-F/R, for NSP4 tyrosinase are 113/114-F/R, for Mefp-5 are 105-F/R. Concentrations: IDT-OMPHT is 11.0 ng/μl, NSP4-Tyrosinase is 16.7 ng/μl, Mefp-5 is 18.2 ng/μl.     |  |  | | --- | --- | | Q5® High-Fidelity 2X Master Mix | 25 ul | | Primer mix (10cM) | 2.5ul | | Template (IDT-OMPHT/NSP4 tyrosinase/Mefp-5) | Xμl (We used 10 ng of the fragment as template for each of them) | | ddH20 | 22.5-X | | Total | 50μl |   PCR setting: Tm for IDT-OMPHT is 68°C, for NSP4 tyrosinase is 62°C, for Mefp-5 is 65°C.   |  |  |  | | --- | --- | --- | | Initial denaturation | 98°C | 30s | | X35 Cycles | 98°C | 10s | | Tm | 20s | | 72°C | 45s | | Final extension | 72°C | 2min | | Hold | 4°C | Infinite | |
| Results | None |

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May 21, 2019
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|  |  |
| --- | --- |
| Experiments | 052019 gBlocks PCR fragments checking via gel running  Repeat gBlocks PCR amplification: IDT-OMPHT,NSP4 tyrosinase,Mefp-5  052191 gBlocks PCR fragments checking via gel running |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gel running: Make two different size gels. The small one is 25ml TAE with 0.25g agarose and 1.25ml SYBR safe dye. This gel is for checking the 052019 PCR fragments. The big one is 50ml TAE with 0.5g agarose and 2.5ml SYBR safe dye. This gel is for checking the 052119 PCR fragments. Mix 5μl PCR product and 1μl 6X loading dye. Run at 130V for 30 min and then check the image.   PCR reaction and setting: The reaction mix is the same as 052019. Only difference we made is that we changed the Tm into three sets in order to figure out whether Tm led to the incorrect bands for image052019. Tm for Set A is 65°C, Tm for set B is 57°C, and Tm for Set C is 53°C.   ![](https://static.igem.org/mediawiki/2019/2/26/T--UM_Macau--PicUploadTesting.png) |
| Results | After rechecking, we found that we mistakenly ordered the primers for NSP4 tyrosinase. Actually the fragment we ordered has already contained the homologous regions that we wanted. |

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May 23, 2019
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|  |  |
| --- | --- |
| Experiments | Restriction digestion of plasmid vectors  Recheck gBlocks NSP4 tyrosinase |
 Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Digestion reaction:   |  |  | | --- | --- | | pBAD24 (102.9 ng/μl) | 30 μl (30mg of DNA) | | EcoRI-HF | 1 μl | | XbaI | 1 μl | | Cut smart | 5 μl | | ddH2O | 13 μl | | Total | 50μl |   Incubate at 37°C overnight   Gel running: Run the PCR products of IDT-OMPHT and Mefp-5, and directly run the gBlocks NSP4 tyrosinase at 130V for 30 min and then check the image. |
| Results | ![](https://static.igem.org/mediawiki/2019/b/b0/T--UM_Macau--Lablog_23_May.png) We got the correct band for NSP4 tyrosinase even though it was very weak. We would try to amplify after ligation. |

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May 24, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gel running: 50 μl digested sample + 10 μl loading dye. The order of loading is shown below. Then separate 60μl into two wells on the gel because the well will be overflowing if we put all 60 μl into one well.   |  |  |  |  | | --- | --- | --- | --- | | 1 kb ladder  (2 μl) | Sample  (30 μl) | Sample  (less than 30 μl) | 1 kb ladder  (2 μl) |   Run at 120V for 23 min in order to separate the digestion products and obtain the target vector (pBAD24, 4516 bp) from unused fragments (26 bp).  Gel purification: We’re using protocol from Illustra GFX PCR DNA and Gel Band Purification Kit Gel. Gel slices weight are 258 mg (used 258 μl CBT3) and 280 mg (used 280 μl CBT3)\*CBT3 = capture buffer type 3  Sample capture → sample binding → wash & dry → elution  We used 1 Microspin column and 1 collection tube for purification of 2 gels. Then Elution with 20μl elution buffer type 6  Gibson Assembly reagents calculation:   |  |  | | --- | --- | | Insert (NSP4 + tyrosinase) | Vector (pBAD24) | | Length: 1004 bp | Length: 4542 bp | | Concentration: 16.7 ng/μl | Concentration: 32.80 ng/μl | | Volume used for ligation: 5 μl | Volume used for ligation: 3 μl | | Mass: 83.5 ng | Mass: 98.4 ng | | Mole: 134.6 fmol / 0.1346 pmol  (by NEBioCalculator) | Mole: 35.06 fmol / 0.03506 pmol  (by NEBioCalculator) |   Volume used (Vector : Insert = 1 : 4)  Gibson assembly reaction:   |  |  | | --- | --- | | Insert (NSP4 + tyrosinase) | 5 μl | | Vector (pBAD24) | 3 μl | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 2 μl | | Total | 20 μl | |
| Results | None |

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May 25, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gibson assembly reaction as negative control for 052419:   |  |  | | --- | --- | | Master Mix | 10 μl | | Digested Plasmid | 2 μl | | ddH2O | 18 μl | | Total | 30 μl |   Incubated at 50°C for 15 minutes.    Transformation of NSP4-Tyrosinase-pBAD24 into competent cells:  a. 50 μl competent cells + 5 μl Gibson assembly mix made on 052419  b. 50 μl competent cells + 5 μl negative control made on 052519  c. Spread on two plates with Ampicillin and incubate at 37°C overnight |
| Results | None |

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May 26, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking and incubation:   |  |  | | --- | --- | | Plate | Colony number | | Negative control | 9 small colonies | | pBAD24-NSP4 tyrosinase | 1 colony |   Prepare the LB Broth with Ampicillin antibiotic and tubes with 1 mL – 5 mL LB Broth with Ampicillin antibiotic.  Pick the only one single colony from the pBAF24-NSP4 tyrosinase transformation plate.  And randomly pick 5 colonies from the negative control transformation plate. Incubate at 37°C overnight. |
| Results | ![](https://static.igem.org/mediawiki/2019/3/3d/T--UM_Macau--Lablog_26_May_1.png) ![](https://static.igem.org/mediawiki/2019/5/57/T--UM_Macau--Lablog_26_May_2.png) ![](https://static.igem.org/mediawiki/2019/1/1d/T--UM_Macau--Lablog_26_May_3.png) The negative control has much more colonies than our pBAD24-NSP4 tyrosinase. So we guessed we might mistakenly record the two plates. The results would show us whether our assumption was true after the plasmid extraction and gel running. |

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May 27, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid extraction:  a. Collect 3 mL of overnight suspension culture from the five negative control tubes.  b. Elute with 50μl elution buffer. Did the elution steps three times with the same 50μl elution buffer. Incubate 4min between each centrifuge.  c. Then use Nanodrop to test the extracted plasmid concentration.    Restriction enzyme digestion reaction: Total 50μl   |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | | DNA (500 ng) | 39.1μl | 27.2μl | 16μl | 18μl | 18μl | | EcoRI-HF | 0.5μl | 0.5μl | 0.5μl | 0.5μl | 0.5μl | | XbaI | 0.5μl | 0.5μl | 0.5μl | 0.5μl | 0.5μl | | Cut smart | 5μl | 5μl | 5μl | 5μl | 5μl | | ddH2O | 4.9μl | 16. 8μl | 28μl | 26μl | 26μl |   Incubate at 37°C overnight |
| Results | DNA plasmid extraction: They are 12.81ng/μl, 18.38ng/μl, 31.41ng/μl, 27.2ng/μl and 28.44ng/μl. |

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May 29, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid identity checking for 052619: Run the digested plasmid from five randomly picked colonies on 052619. Loading order for the gel: Left to right 1 kb ladder -> Colony 1 -> Colony 2 -> Colony 3 -> Colony 4 -> Colony 5   Repeat transformation of bacteria: 5 μl Gibson assembly mix made on 052419 and 5 μl negative control made on 052519. Spread on two plates with Ampicillin and incubate at 37°C overnight. |
| Results | ![](https://static.igem.org/mediawiki/2019/d/d4/T--UM_Macau--Lablog_29_May.jpg) Besides the result of colony 3 that we don’t know how to explain it. Its band is neither the vector nor the NSP4 tyrosinase which was 1022 bp, but this band is around 1500 bp. All the other bands are our vectors which is pBAD24. However, they do not have the insert so they are not the proper plasmid. |

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May 30, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking and incubation   |  |  | | --- | --- | | Plate | Colony number | | Negative control | 3 small colonies | | pBAD24-NSP4 tyrosinase | 2 colonies |   Pick the two colonies from the pBAF24-NSP4 tyrosinase transformation plate.  And pick the 3 colonies from the negative control transformation plate. Incubate at 37°C overnight. |
| Results | ![](https://static.igem.org/mediawiki/2019/a/a8/T--UM_Macau--Lablog_30_May_1.png)  ![](https://static.igem.org/mediawiki/2019/d/de/T--UM_Macau--Lablog_30_May_2.png)  ![](https://static.igem.org/mediawiki/2019/8/8c/T--UM_Macau--Lablog_30_May_3.png)  ![](https://static.igem.org/mediawiki/2019/b/bb/T--UM_Macau--Lablog_30_May_4.png)  ![](https://static.igem.org/mediawiki/2019/0/05/T--UM_Macau--Lablog_30_May_5.png)  ![](https://static.igem.org/mediawiki/2019/3/3e/T--UM_Macau--Lablog_30_May_6.png) |

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May 31, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid extraction:  a. Collect 3 ml of overnight suspension culture from the two pBAD24-NSP4 tyrosinase tubes.  b. Elute with 50μl elution buffer. Did the elution steps three times with the same 50μl elution buffer. Incubate 4min between each centrifuge.  c. Then use Nanodrop to test the extracted plasmid concentration. They are 46ng/μl and 28ng/μl.    Restriction enzyme digestion reaction: Total 25μl   |  |  |  | | --- | --- | --- | | DNA (500 ng) | 15μl | 18μl | | EcoRI-HF | 0.5μl | 0.5μl | | XbaI | 0.5μl | 0.5μl | | Cut smart | 2.5μl | 2.5μl | | ddH2O | 10.5μl | 3.5μl |   Incubate at 37°C overnight |
| Results | None |

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# June

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June 1, 2019
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|  |  |
| --- | --- |
| Experiments | Tyrosinase secretion system construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid identity checking for 053019: Run the digested plasmid from two picked colonies from pBAD24-NSP4 tyrosinase plate on 053019 at 120V for 25 min. Loading order is 1 kb ladder, sample1, sample2, 1 kb ladder. Load 25μl digestion product with 5μl loading dye. |
| Results | ![](https://static.igem.org/mediawiki/2019/8/8d/T--UM_Macau--Lablog_1_Jun.jpg) No correct colonies. Through these two transformation experiments, we thought our competent cells have some problem. So we would change to use a new one. |

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June 3, 2019
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|  |  |
| --- | --- |
| Experiments | Vector identities checking via gel running |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Expected band size for each plasmid backbone:  pBBR1MCS2 = 5148 bp and pBAD24 = 4542 bp   I: First gel running around 10 am by Hugh  Samples loaded :  10μl pBBR1MCS-2 + 2μl loading dye (x6)  10μl pBAD24 + 2μl loading dye (x6)   Ran for 120 V for 25 minutes. Initial observation was that the samples were floating and not settling down even after the addition of the loading dye. Hugh did not run this gel and prepared a new one instead because the TA said maybe the gel preparation is not good.   II: Second gel running around 11 am by Hugh  Samples loaded:  10μl pBBR1MCS-2 + 2μl loading dye (x6)  10μl pBAD24 + 2μl loading dye (x6)   Ran for 120 V for 25 minutes. Gel image is figure 1.   III: Third gel running around 5 pm by Philip  Samples loaded:  24μl pBBR1MCS-2 + 20μl loading dye (x6)  24μl pBAD24 + 20μl loading dye (x6)   Ran for 120 V for 25 minutes. Initial observation: Even after mixing the large volume of loading dye. When loading the sample it was still floating away. We now suspected that there might be something wrong with the sample. We think the sample contains ethanol. Gel image is figure 2.   IV: Fourth gel running around 6 pm by Philip  Samples loaded:  10μl pBBR1MCS-2 + 2μl loading dye (x6)  10μl pBAD24 + 2μl loading dye (x6)   Ran for 120 V for 25 minutes. Initial observation: Same thing as the previous gel   No correct band at all, I think it’s due to the loading dye making the fragments too heavy and thus it did not run fast in the gel, therefore our Xiao Xiong (One of our TAs)  recommended to prepare a new gel and use a smaller well size. After this result, Xiao Xiong has put the stock plasmid backbone tubes onto a heating block; she opened the cap of the tubes and heated the tubes at 55 C, I reclaimed the stuff after maybe 30 minutes; this was to evaporate possible ethanol contaminants. |
| Results | ![](https://static.igem.org/mediawiki/2019/1/11/T--UM_Macau--Lablog_3_Jun_1.png) Sample might have floated away and none were actually run in the gel.     ![](https://static.igem.org/mediawiki/2019/d/d3/T--UM_Macau--Lablog_3_Jun_2.png) No correct band at all, I think it’s due to the loading dye making the fragments too heavy and thus it did not run fast in the gel, therefore our Xiao Xiong (One of our TAs) recommended to prepare a new gel and use a smaller well size. After this result, Xiao Xiong has put the stock plasmid backbone tubes onto a heating block; she opened the cap of the tubes and heated the tubes at 55 C, I reclaimed the stuff after maybe 30 minutes; this was to evaporate possible ethanol contaminants.     ![](https://static.igem.org/mediawiki/2019/8/83/T--UM_Macau--Lablog_3_Jun_3.png) It seems that the band is still not at the correct place. There is not even a trace of the correct band for pBAD24 it seems like the band is located more than 10kb. For pBBR1MCS-2, it seems like the DNA sample all floated away and what we saw that settled were probably just the loading dye. Next time we should mix the loading dye and the DNA sample better.     ![](https://static.igem.org/mediawiki/2019/9/97/T--UM_Macau--Lablog_3_Jun_4.jpg) Maybe there is something wrong with the plasmid? After consultation with Professor Zheng Jun’s PhD. Student, apparently we should first digest the plasmid vector with at least one enzyme to linearize it. Only then should we run it in the gel and check the size. Apparently if we do not linearize the plasmid before running, due to its circular form it would run slower along the gel causing it to show a non-true band. |

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June 4, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat vector identities checking via gel running |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Restriction enzyme digestion reaction for vectors: Total 10μl A1 is pBBR1MCS-2 041719, A2 is pBBR1MCS-2 050519, B1 is pBAD24 041719, B2 is pBAD24 050519.    |  |  |  |  |  | | --- | --- | --- | --- | --- | | Vector | A1 | A2 | A3 | A4 | | DNA (500 ng) | 4μl | 8.5μl | 1μl | 5μl | | EcoRI-HF | 0.5μl | 0.5μl | 0.5μl | 0.5μl | | Cut Smart | 1μl | 1μl | 1μl | 1μl | | ddH20 | 4.5μl | 0μl | 7.5μl | 3.5μl |    Incubate at 37℃ for 1h. Gel running: 120V for 25 min. 10μl digestion product + 2μl loading dye. Gel arrangement is 1kb ladder, A1, B1, B2, A2, 1 kb ladder. Restriction enzyme digestion reaction for insert: Total 50μl. Incubate overnight.    |  |  | | --- | --- | | Insert DNA (200 ng) | 8.17μl | | EcoRI-HF | 2μl | | Xbal | 2ul | | ddH20 | 37.83μl | | Cut Smart | 5μl | |
| Results | ![](https://static.igem.org/mediawiki/2019/7/7b/T--UM_Macau--Lablog_4_Jun.jpg)   After digestion with EcoRI-HF for 1 hr at 37 C. We ran the plasmid stock that has kept (041719) along with the stock plasmid solution that has prepared (050519). The 041719 are at the expected size: pBBR1MCS2 = 5148 bp and pBAD24 = 4542 bp. |

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June 5, 2019
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|  |  |
| --- | --- |
| Experiments | Vector and insert checking via gel running |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Sample:55μl digested insert + 10μl loading dye  Order: 1 kb ladder(μl), sample(30μl), sample(30μl), 1 kb ladder(2μl)  Run at 120V for 30 min.  Our expected size is 4515bp for vector pBAD24 and 1004bp for NSP4+tyrosinase |
| Results | ![](https://static.igem.org/mediawiki/2019/7/72/T--UM_Macau--Lablog_5_Jun.jpg)   The left one is for vector pBAD24 and the other one is for insert NSP4+tyrosinase. The right one was the supposedly digested insert but we did not see any band there. |

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June 6, 2019
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|  |  |
| --- | --- |
| Experiments | pBAD24, pBBR1MCS2 plasmid extraction for sequencing |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Pellet 4 ml of the 5 ml overnight culture. Pipette 1 ml into microcentrifuge tube and centrifuge for 3 min. Discard supernatant and add next 1 ml into same tube. Repeat until you’ve pelleted 4 ml worth of bacteria culture. (12 min of centrifugation)  Follow protocol in QIAGEN (miniprep) plasmid extraction kit.   \*Miniprep is for 5 ml bacteria culture, yield is low because starting vol. is low.      Mediprep is for 50 ml, maxi for 100, and giga for 1L.  P1 is resuspension buffer.  P2 is lysis buffer. (Alkaline, with many salts)  N3 is neutralization buffer. (Balance alkalinity)  You’d see some white precipitate after adding N3. The centrifuge step in protocol is to spin down most of that ppt. for you to collect supernatant. This prevents clogging (blocking) of the spin column for downstream procedure.  Nanodrop at Prof. Xu’s Lab. Clean with water and blank, then measure.  pBAD24: 66.4ng/μl (acceptable)  pBBR1MCS2: 37.6ng/μl (still pretty low)  Afternoon: Philip sent them for plasmid sequencing. |
| Results | None |

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June 14, 2019
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|  |  |
| --- | --- |
| Experiments | Resuspension of gBlock fragments |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Centrifuged the tube at 4500 x g to make sure the dried pellet is at the bottom of the tube. Added 100μl TE buffer to all of the fragments (I was expecting to get 10ng/μl). All products that were delivered were 1000 ng. Vortexed briefly. Incubated at 50 C for 20 minutes. Vortexed then centrifuged. Got the concentration using Nanodrop, we used TE buffer as blank. After the last step, I aliquoted 50μl to another tube so that we would avoid exposing the main stock to many freeze/thaw cycles which could damage the DNA too. So there is about 50μl fragment volume left on the main IDT tubes that we have received. |
| Results | Resulting concentration:   |  |  |  | | --- | --- | --- | | Concentration | Aliquoted tube (50μl) | IDT Tube (~50μl) | | NSP4-Tyrosinase   (1022 bp) | 10.84 ng/μl = 0.017 pmol | 8.341 ng/μl = 0.013 pmol | | OPHT (1495 bp) | 8.82 ng/μl = 0.0095 pmol | 7.147 ng/μl = 0.0077 pmol | | OPHTII (2211 bp) | 7.65 ng/μl = 0.0055 pmol | 6.968 ng/μl = 0.0075 pmol | | OPHTIII (1507 bp) | 5.68 ng/μl = 0.0060 pmol | 23.29 ng/μl = 0.02501 pmol | | OPHM (1782 bp) | 3.85 ng/μl = 0.00349 pmol | 5.164 ng/μl = 0.0047 pmol |   Pmol calculations through <https://nebiocalculator.neb.com/#!/dsdnaamt>. |

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June 15, 2019
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|  |  |
| --- | --- |
| Experiments | Restriction digestion of pBBR1MCS2 |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Restriction digestion reaction: (pBBR1MCS2 concentration:75.5ng/μl)   |  |  | | --- | --- | | pBBR1MCS2(2.5mg) | 2500/75.5≈33ml | | EcoRI | 3μl | | BamHI | 3μl | | Cut smart | 5μl | | dd H2O | 6μl | | Total | 50μl |   Put in thermocycler for 1h. Settings are as following: 37℃ for 1h, 4℃ for infinite  Gel running: Loading samples (50μl+10 loading dye). Put original plasmid to compare with digested one. Run at 120V for 20 min.  Gel purification: After excise bands, we did gel purification using protocol from Illustra GFX PCR DNA & Gel Band Purification Kit Gel. Final elution volume is 40μl. Then using Nanodrop to check its concentration which is 7.278ng/μl. It is not a good concentration result. So we repeated the experiment again.  Repeat restriction digestion: Prepare 4 tubes, 50μl each, total 200μl. The reaction is the same as previous. Overnight digestion at 37℃. |
| Results | ![](https://static.igem.org/mediawiki/2019/2/29/T--UM_Macau--Lablog_15_Jun.jpg)   Both are the digested products, we just separated them into two wells like usual because one well might overflow and we would lose DNA. Both of them pBBR1MCS2 are at the expected size. |

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June 16, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat gel purification |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gel running: This time we put all digested products into one big well. (200μl sample+40μl loading dye). Order is 1kb ladder, sample, and 1 kb ladder. Then Still run at 120V for 20 min.    Gel purification: After excising the gel and following the same protocol, we eluted it with 20μl elution buffer. |
| Results | ![](https://static.igem.org/mediawiki/2019/f/fb/T--UM_Macau--Lablog_16_Jun.jpg) |

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June 18, 2019
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|  |  |
| --- | --- |
| Experiments | pBBR1MCS2+OPHTIII construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gibson assembly:   |  |  | | --- | --- | | Insert (OPHT III) | Vector (pBBR1MCS2) | | Length: 1507 bp | Length: 5148 bp | | Concentration: 23.29 ng/μl | Concentration: 52.3 ng/μl | | Volume used for ligation: 3.6 μl | Volume used for ligation: 1.91 μl | | Mass: 83.5 ng | Mass: 98.4 ng | | Mole: 25.01fmol /0.025 pmol  (by NEBioCalculator) | Mole: 16.44 fmol / 0.016 pmol  (by NEBioCalculator) |   Volume used (Vector : Insert = 1 : 3)  Gibson assembly reaction:   |  |  | | --- | --- | | Insert (OPHT III) | 3.6μl | | Vector (pBBR1MCS2) | 1.91μl | | Gibson Assembly Master Mix (2X) | 10μl | | Deionized H2O | 4.49μl | | Total | 20 μl |   Incubate at 50℃ for 1h.  Transformation of bacteria: The only changes that we usually make are the following:   1. Incubation times for both thawing and incubating with DNA. Varies from 25-30 mins 2. The amount of LB broth to put for the outgrowth step varies from 500-900 ul (This case Hugh used 500 ul) 3. Incubation time for the outgrowth step varies from 45 mins - 1 hr (This time we used 1 hr) 4. Centrifuge 3000 x g for 2 minutes after outgrowth step to pellet the bacteria then remove some supernatant to leave 250 ul LB solution then resuspend the pellet with the leftover 250 ul LB solution then plate all the bacteria on the plate. 5. Dry the plates for 10 minutes before inverting and putting into the 37 C incubator |
| Results | None |

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June 19, 2019
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|  |  |
| --- | --- |
| Experiments | pBBR1MCS2+OPHTIII construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking and incubation   1. Picked 6 colonies from the plate 2. Put 8 mL LB broth total and added 8 uL Ampicillin 3. Put in 37 C shaking incubator overnight for 12-16 hrs |
| Results | None |

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June 20, 2019
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|  |  |
| --- | --- |
| Experiments | pBBR1MCS2+OPHTIII construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid extraction: using QIAprep Spin Miniprep Kit.  6 tubes containing 8ml transformed bacteria cultured with LB broth. Pellet 6ml bacterial overnight culture from each tube. Add 250μl buffer D1, then 250μl buffer D2, 350μl buffer N3. Centrifuge at 13000 rpm for 10 min. Transform the 800μl supernatant into spin column. Centrifuge at 13000 rpm for 60s and discard flow through. Add 500μl buffer PB with ethanol. Then centrifuge again at 13000 rpm for 60s and discard flow through. Add 750μl buffer PE, centrifuge at 13000 rpm for 60s and repeat twice. Put the spin columns into new tubes and add 35μl buffer EB. Stand for 5min then centrifuge again. Using Nanodrop to measure their concentration. |
| Results | None |

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June 26, 2019
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|  |  |
| --- | --- |
| Experiments | pBBR1MCS2+OPHTII Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gibson assembly  Transformation of bacteria |
| Results | Failed: subsequent days were used as diagnostic and review |

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June 27, 2019
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|  |  |
| --- | --- |
| Experiments | pBBR1MCS2+OPHTII Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking and overnight culture |
| Results | None |

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June 28, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat pBBR1MCS2+OPHTII Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gibson assembly  Transformation of bacteria |
| Results | None |

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June 29, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat pBBR1MCS2+OPHTII Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid Extraction of pBBR1MCS2: using Midi Kit  Transfer the overnight culture into four 50ml tubes.  Nanodrop result is 54.42 ng/ul    Restriction digestion  PCR for 2 hours at 37 degrees, infinite hold at 4 degree    Gel run for 25 mins at 130V  (We didnt save the gel image results), we cut the lower band and did gel purification for the pBBR1MCS2 vector. |
| Results | None |

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June 30, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat pBBR1MCS2+OPHTII Construction, Bacteria Transformation |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gibson Assembly of pBBR1MCS2 and OPHT2  Run for 1 hour at 50 degrees, infinite hold at 4 degrees    Transformation:  We thawed the competent cells for only 12 minutes, we should thaw them for a longer time. Then we spread the bacteria on Kan+ agar plates, culture them for 16 hours. |
| Results | Transformation: It has no colony on positive control plate but few colonies on OPHT2 plate.  ![](https://static.igem.org/mediawiki/2019/b/ba/T--UM_Macau--Lablog_30_Jun_1.jpg)  ![](https://static.igem.org/mediawiki/2019/1/15/T--UM_Macau--Lablog_30_Jun_2.jpg) |

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# July

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July 1, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat pBBR1MCS2+OPHTII Construction, Start OPHM construction and Transformation of OPHM |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking and bacteria incubation: Picked 4 colonies from OPHTII agar plate for overnight LB broth culture LB broth volume 40ml + Kanamycin volume 0.04ml    Gibson assembly for pBBR1MCS2+OPHM construction:  (insert) OPHM concentration = 5.164ng/ul = 0.0047pmol  (vector) digested pBBR1MCS2 = 12.60ng/ul = 0.004pmol    60ng of vector = 60/12.60 = 4.76ul  4.76ul x 0.004pmol = 0.019 pmol  vector:insert=1:2  Insert = 0.019pmol x 2 =0.038pmol  Volume for insert = 0.038/0.0047 = 8.09ul     2. pBBR1MCS2+OPHM (vector:insert=1:2)  |  |  | | --- | --- | | Insert (OPHM) | 8.09 μl | | 60ng Vector (pBBR1MCS2) | 4.76 μl | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 0 μl | | Total | 22.85 μl |      2. Positive control  |  |  | | --- | --- | | Insert | 0 μl | | Positive control | 10ul | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 0 μl | | Total | 20 μl |      3. Negative control  |  |  | | --- | --- | | Insert | 0 μl | | Vector (pBBR1MCS2) | 10ul | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 0 μl | | Total | 20 μl |   Incubate at 50℃ for 1h.    Transformation:  a. Heat shock at 42℃ for 45 secs using water bath.  b. Incubate at 37℃ for 60 mins, then plated them into three plates and cultured them for 16 hours. |
| Results | Transformation: The positive control plate has no colony due to our mistake of plating the bacteria to Kan+ agar plate, it should be plated on Amp+ agar plate.The negative control plate has many colonies which should not happen, we suspect that the plasmid is self-ligated although we transformed with vector without insert. The OPHM agar plate has man colonies which is the expected results. |

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July 2, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat pBBR1MCS2+OPHTII Construction  Plasmid Extraction of pBBR1MCS2-OPHT2  Restriction digestion for plasmid identity checking |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid Extraction: We eluted with 40ul elution buffer for x3 times.    Restriction digestion reaction:   |  |  |  |  |  | | --- | --- | --- | --- | --- | |  | C1 | C2 | C3 | C4 | | 500ng DNA | 500/82.28=6.1ul | 500/87.78=5.7ul | 500/85.39=5.9ul | 500/88.74=5.6ul | | EcoRI | 0.5μl | 0.5μl | 0.5μl | 0.5μl | | BamHI | 0.5μl | 0.5μl | 0.5μl | 0.5μl | | Cut smart | 1μl | 1μl | 1μl | 1μl | | dd H2O | 1.9μl | 2.3μl | 2.1μl | 2.4μl | | Total | 10μl | 10μl | 10μl | 10μl |   Put in thermocycler for 2hrs. Settings are as following: 37℃ for 2hrs, 4℃ for infinite hold.  Gel running: Load 4 samples (10μl+2 loading dye) and 1kb ladder (2ul) into wells. Put original plasmid to compare with digested one. Run at 120V for 20 min.    Restriction digestion reaction: (pBBR1MCS2 concentration:75.5ng/μl)   |  |  | | --- | --- | | pBBR1MCS2(2ug) | 40ul | | EcoRI | 1μl | | BamHI | 1μl | | Cut smart | 5μl | | dd H2O | 3μl | | Total | 50μl |   We made two tubes of the reaction above. Put in thermocycler for 14hrs. Settings are as following: 37℃ for 14hrs, 4℃ for infinite. |
| Results | Concentration of OPHT2 plasmid: colony 1(C1)=82.28ng/ul  colony 2(C2)=87.78ng/ul  colony 3(C3)=85.39ng/ul  colony 4(C4)=88.74ng/ul   Gel run: The expected results should be two bands shown on the gel as the plasmids should be digested by enzyme at the restriction sites. However, there was only a band for each sample, it might due to the plasmid is undigested or not fully digested. Therefore, we have to reconstruct the plasmid backbone. |

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July 3, 2019
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|  |  |
| --- | --- |
| Experiments | Repeat pBBR1MCS2+OPHTII Construction  pBBR1MCS2+OPHM Construction  Prepare LB agar plates |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Gel purification: Run gel. Order is 1kb ladder, pBBR1MCS2, pBBR1MCS2, none, OPHT II, pBBR1MCS2, pBBR1MCS2. Used 20μl elution buffer type 6. Concentration is 25.42ng/μl.   Gibson assembly reaction:  OPHT II concentration: 7.645ng/μl = 0.0055pmol  pBBR1MCS2 concentration: 25.42ng/μl = 0.008pmol  OPHM concentration: 5.164ng/μl = 0.0047pmol  1.pBBR1MCS2+OPHTII (insert: vector=3:1)   |  |  | | --- | --- | | Insert (OPHT II) | 8.7 μl | | 100 ng Vector (pBBR1MCS2) | 3 μl | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 0 μl | | Total | 21.7 μl |  2. pBBR1MCS2+OPHM (insert: vector=2:1)  |  |  | | --- | --- | | 100 ng Insert (OPHM) | 10.21 μl | | Vector (pBBR1MCS2) | 3 μl | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 0 μl | | Total | 23.2 μl |  3. Negative control  |  |  | | --- | --- | | Insert | 0 μl | | Vector (pBBR1MCS2) | 10ul? | | Gibson Assembly Master Mix (2X) | 10 μl | | Deionized H2O | 0 μl | | Total | 20 μl |   Incubate at 50℃ for 1h.    Transformation of OPHT2, OPHM and negative control:  a. Heat shock at 42℃ for 45secs using water bath.  b. Incubate at 37℃ for 45 min then plated them into three plates.  c. Culture them for 12 hrs.    Prepare LB agar plates: 200ml LB agar= 8g powder+ 200ml MilliQ water.  The ratio for antibiotics and solution is 1:1000. So 200μl Amp/Kan to each kind of plate.  We made totally 18 plates with Kan+ and 21 plates with Amp+. |
| Results | None |

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July 4, 2019
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|  |  |
| --- | --- |
| Experiments | Colony picking for OPHT2, OPHM Transformation of OPHT1, OPHT3 and NSP4-Tyrosinase  Amplify the plasmid  Plasmid Extraction of OPHT2, OPHM  Restriction digestion to check plasmid identity |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking: We picked 6 colonies from each transformed plasmids and incubate them in 5mL LB with antibiotic for 12-14 hrs.   Transformation: Transform OPHT2(#2),  OPHT3(#2), NSP4(#6) into Dh5 strain E.coli and incubate at 37 degrees.   Amplify the plasmid by streaking bacteria glycerol stock and culture in 5mL LB broth with antibiotic.   Plasmid Extraction: Save overnight culture before extraction. Used 30ul elution buffer and eluted 3 times, each time incubated 3 minutes. Concentration of DNA is measured by using Nanodrop.    Restriction digestion reaction:   |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | |  | #1 | #2 | #3 | #4 | #5 | #6 | | 500ng OPHM | 500/95.65=5.3ul | 5.3ul | 5.3ul | 4.8ul | 5.1ul | 5.5ul | | EcoRI | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | | BamHI | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | | Cut smart | 1μl | 1μl | 1μl | 1μl | 1μl | 1μl | | dd H2O | 3.1μl | 3.1μl | 3.1μl | 3.6μl | 3.3ul | 2.9ul | | Total | 10μl | 10μl | 10μl | 10μl | 10μl | 10μl |        |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | |  | #1 | #2 | #3 | #4 | #5 | #6 | | 500ng OPHT2 | 4.3ul | 4.1ul | 5.2ul | 4.6ul | 4.7ul | 4.9ul | | EcoRI | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | | BamHI | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | 0.3μl | | Cut smart | 1μl | 1μl | 1μl | 1μl | 1μl | 1μl | | dd H2O | 4.1ul | 4.3ul | 3.2ul | 3.8ul | 3.7ul | 3.5ul | | Total | 10μl | 10μl | 10μl | 10μl | 10μl | 10μl |     Put in thermocycler for 4hrs. Settings are as following: 37℃ for 4hrs, 4℃ for infinite hold.    Run the samples on the agarose gel and observe the DNA bands under UV. The estimated correct band size of plasmid colonies are sent for RNA sequencing. |
| Results | Concentration of DNA:  OPHM                                 OPHT2  #1=95.65ng/ul                   #1=117.1ng/ul  #2=94.81ng/ul                   #2=124.3ng/ul  #3=94.47ng/ul                   #3=97.22ng/ul  #4=204.6ng/ul                   #4=109.4ng/ul  #5=98.68ng/ul                   #5=108.2ng/ul  #6=91.51ng/ul                   #6=102.3ng/ul    Gel run and observation:  OPHT2 (#2 ans #5)  OPHM(#2 and #6) |

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July 10, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | 1.Preparation of 20% Arabinose  2.SDS-Dye preparation  3.Protein samples preparations |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1: Prepared 5 mL 20% arabinose  Mixed 1051.9 mg Arabinose + 5 mL milliQ water   2: 4x SDS sdye preparation   |  |  | | --- | --- | | **Component name** | **Volume** | | 4 x stacking gel buffer | 25 mL | | SDS | 2 g | | Glycerol | 20 mL | | Bromophenol blue 2-mercaptoethanol | 20 mg |   3: Protein samples preparation   OPHT, OPHTIIII and NSP4 Pellets  -Resuspended them all with 75 ul Milli-Q water + 25 ul 4 x SDS Page dye. Stored in -20C fridge  Stored 1 mL of the supernatant of the induced culture tube for later protein expression test.  Prepared loading sample for supernatant. 75 ul NSP4 supernatant + 25 ul 4x SDS page dye |
| Results | None |

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July 11, 2019
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|  |  |
| --- | --- |
| Performers | Renee |
| Experiments | pBBR1MCS2+OPHTII(sequenced) Construction  pBBR1MCS2+OPHM(sequenced) Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Transformation:  2.5ul OPHTII #5 with 50ul competent cell  2ul OPHM #2 with 50ul competent cell  Plated at 16:15 and incubated at 37 degree overnight. |
| Results | None |

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July 12, 2019
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| --- | --- |
| Performers | Philip |
| Experiments | SDS-Page gel running and Coomassie blue staining |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Gel percentage used 12.5%  Protein samples that were ran:  NSP4 = 37.17 kDA  OPHTI = 52.18 kDA  OPHTIII = 52.83 kDA    10x Prepared running buffer:   |  |  | | --- | --- | | **Component name** | **Amount** | | Tris | 30g | | Glycine | 144g | | SDS | 10g |     Gel running arrangement:  NSP4 supernatant -> NSP4 pellet -> OPHT3 -> OPHT -> Ladder -> NSP4 supernatant -> NSP4 pellet -> OPHT3 -> OPHT -> Ladder  Running time: 60V for 1hr then 120 V for 1 hr    Coomasie blue staining:   1. Washed the SDS away from the gel 2. Soaked the gel with the gel stain for 1 hr 3. Wash the excess gel stain with dH2O. 4. Put water after 3 times washing, boil for 5 minutes in the microwave then let it shake overnight. Start time 10:27 pm 5. Remove the water and take picture |
| Results | None |

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July 16, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Renee |
| Experiments | Make glycerol stock of pBBR1MCS2+OPHTII(sequenced) and pBBR1MCS2+OPHM (sequenced) |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 200ul 5% glycerol + 800ul bacteria culture   Separate 10 tubes SDS dye |
| Results | None |

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July 19, 2019
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|  |  |
| --- | --- |
| Performers | Renee |
| Experiments | Test adhesion system in three cell lines |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Transformation:  TOP10           OPHT#1(1ul)     OPHT3 #3(1ul)     07/05 vector (3.5ul)  BL-21            OPHT#1(1ul)     OPHT3 #3(1ul)     07/05 vector (3.5ul)  MG1655        OPHT#1 (1ul)     OPHT3 #3(1ul)     07/05 vector (3.5ul) |
| Results | None |

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July 20, 2019
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|  |  |
| --- | --- |
| Performers | Renee |
| Experiments | Test adhesion system in three cell lines |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Pick colony: three colonies per plate for TOP10 and BL-21 with OPHT, OPHT3 and vector, totally 18 tubes, culture at 37 degree shaker overnight  Transformation:  TOP10          OPHT2       OPHM  BL-21           OPHT2       OPHM  MG1655       OPHT2       OPHM |
| Results | No colony grew on all MG1655 plates. Repeat the plating with all the remainder MG1655 culture. |

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July 21, 2019
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|  |  |
| --- | --- |
| Performers | Renee |
| Experiments | Test adhesion system in three cell lines |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Pick colony: three colonies per plate for TOP10 and BL-21 with OPHT2 and OPHM, totally 12 tubes, culture at 37 degree shaker overnight |
| Results | Still no colony grew on all MG1655 plates. MG1655 was not suitable for our expression system. |

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August 9, 2019
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|  |  |
| --- | --- |
| Performers | Renee, Philip |
| Experiments | pet11a+OPHT/OPHT2/OPHT3/OPHM/OPHA1/OPHA2 Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1. Gel making 2. Gel running: 50ul PCR product + 10ul loading dye, load 30ul per well for pet11a+OPHT/OPHT2/OPHT3/OPHM/OPHA1/OPHA2; 3x50ul PCR product + 30ul loading dye, load 90ul per well for pet11a vector. Run at 120V 15mins   Loading order: Gel1-ladder/OPHT1/OPHT1/OPHT1/OPHT2/OPHT2                             Gel2-blank/ladder/OPHT3/OPHT3/OPHM/OPHM                             Gel3-ladder/OPHA1/OPHA1/blank/OPHA2/OPHA2                             Gel4-ladder/vector/vector    Gel cut weight: OPHT1     0.36g           Buffer type 3: 360ul                              OPHT2     0.27g                                     270ul                              OPHT3     0.31g                                     310ul                              OPHM      0.31g                                     310ul                              OPHA1     0.16g                                     160ul                              Vector1    0.57g                                     570ul                               Vector2   0.43g                                     430ul    Concentration: OPHT1   41ng/ul                              OPHT2   38.36ng/ul                              OPHT3   18.95ng/ul                              OPHM   21.67ng/ul                              OPHA1  10.02ng/ul                              Vector1+ Vector2  54.12ng/ul |
| Results | No band for OPHA2 |

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August 10, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee |
| Experiments | Repeat pet11a-OPHA2 Construction  Continue pet11a-OPHT/OPHT2/OPHT3/OPHM/OPHA1 Construction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1. OPHA2 PCR followed by running gel  2. Gibson assembly:   |  |  | | --- | --- | | OPHT1 | 1.91ul | | pet11a vector (100ng) | 1.85ul | | H2O | 6.24ul |      |  |  | | --- | --- | | OPHT2 | 3ul | | pet11a vector (100ng) | 1.85ul | | H2O | 5.15ul |      |  |  | | --- | --- | | OPHT3 | 4.2ul | | pet11a vector (100ng) | 1.85ul | | H2O | 3.95ul |      |  |  | | --- | --- | | OPHM | 4.2ul | | pet11a vector (100ng) | 1.85ul | | H2O | 3.95ul |      |  |  | | --- | --- | | OPHA1 | 7.63ul | | pet11a vector (100ng) | 1.85ul | | H2O | 0.52ul |   3. Transformation: 2ul DNA+50ul competent cell Stbl3 Transformed OPHT1, OPHT2, OPHT3, OPHM, OPHA1 and positive ctrl; plated 300ul transformed bacteria on agar plates |
| Results | Still no band showed on the gel for OPHA2 |

Close

August 12, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Plasmid Extraction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Used 30ul elution buffer for elution |
| Results | DNA Concentration (ng/ul):     |  |  | | --- | --- | | OPHT #1 | 109.3 | | OPHT #2 | 156.8 | | OPHT #3 | 116.4 | | OPHT2 #1 | 124.6 | | OPHT2 #2 | 116.5 | | OPHT2 #3 | 147.4 | | OPHT3 #1 | 107.9 | | OPHT3 #2 | 105.8 | | OPHT3 #3 | 140.9 | | OPHM #1 | 148.0 | | OPHM #2 | 94.47 | | OPHM #3 | 118.0 | | OPHA #1 | 126.6 | | OPHA #2 | 83.39 | | OPHA #3 | 114.0 | |

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August 23, 2019
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| --- | --- |
| Performers | Philip, Julien |
| Experiments | Tyrosinase Activity Assay |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Sample 1: Induced NSP4 pellet Sample 2: Induced NSP4 supernatant  Sample 3: Uninduced NSP4 pellet  Sample 4: Uninduced NSP4 supernatant    Homogenize cells with 500ul ice-cold Tyrosinase Assay buffer to perform lysis and keep on ice for 10 minutes followed by centrifugation at 10000xg for 15 minutes at 4 degree. Collect the supernatant and estimate protein concentration by using BCA protein assay kit (protein concentration should range between 1 to 2.5 ug/ul) |
| Results | None |

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# September

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September 3, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee |
| Experiments | Prepare NSP4 for Negative control  NSP4 activity test preparation |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | 1. Took one tip of cells from glycerol stock, incubate with 5ml LB broth + 5ul Ampicillin at 37 degree shaker at 17:05 2. Pull down assay for NSP4 induced supernatant. 3. Sonication for NSP4 bacteria culture |
| Results | None |

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September 8, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Nickel pull-down assay  Tyrosinase activity test |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | 1. Prepare nickel resin: Pipette 30ul of nickel resin. Wash resin in 1ul H2O two times followed by washing with 1ml binding buffer two times. 2. Pull-down assay: Centrifuge the induced BL-21 (NSP4 transformed colony 3) as well as the uninduced one and take the supernatant as pull-down assay sample. Pipette 30ul resuspend resin into supernatant. Incubate and rotate it at 4 degree for  45mins. Spin at 3900rpm for 10mins, remove the supernatant which will be stored just in case, then leave 1ml fraction in  resuspend resin and transfer resin to  1.5ml tube. Spin down at 4000g 2min to get resin. Discard supernatant. Wash resin with 750ul binding buffer three times (we washed the resin with Tyrosinase Assay buffer in the third wash). Measure protein concentrationProceed to Tyrosinase Activity Assay. 3. Tyrosinase activity test: Homogenize cells with 500ul ice-cold Tyrosinase Assay buffer to perform lysis and keep on ice for 10 minutes followed by centrifugation at 10000xg for 15 minutes at 4 degree. Collect the supernatant and estimate protein concentration by using BCA protein assay kit (protein concentration should range between 1 to 2.5 ug/ul) |
| Results | |  |  | | --- | --- | | Protein | Concentration | | Uninduced pellet | 1.67ug/ul | | Induced pellet | 5.64 ug/ul | | Uninduced supernatant | -0.19ug/ul | | Induced supernatant | -0.11ug/ul |   As the induced supernatant concentration is negative, there is literally no protein in the supernatant, therefore we did not continue proceed the Tyrosinase Activity test.   ![](https://static.igem.org/mediawiki/2019/f/f2/T--UM_Macau--Lablog_8_Sep_1.jpg)  ![](https://static.igem.org/mediawiki/2019/7/77/T--UM_Macau--Lablog_8_Sep_2.jpg)  ![](https://static.igem.org/mediawiki/2019/7/71/T--UM_Macau--Lablog_8_Sep_3.jpg)  ![](https://static.igem.org/mediawiki/2019/b/b1/T--UM_Macau--Lablog_8_Sep_4.jpg)  ![](https://static.igem.org/mediawiki/2019/3/31/T--UM_Macau--Lablog_8_Sep_5.jpg)  ![]() |

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September 9, 2019
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|  |  |
| --- | --- |
| Performers | Julien |
| Experiments | Make an overnight culture of BL-21 (NSP4 transformed colony 3) from glycerol stock |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Make a 20mL culture of BL-21 glycerol stock (NSP4 transformed colony 3) with Ampicillin antibody (1:1000 dilution) and start incubation in 37 degree shaking incubator at 6.25pm |
| Results | None |

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September 10, 2019
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|  |  |
| --- | --- |
| Performers | Julien, Renee |
| Experiments | pBAD24 NSP4 #3 transformation with BL-21  Induce overnight BL-21 (NSP4 transformed colony 3) |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1. Transformation: 1.5ul pBAD24 NSP4 #3 + 50ul BL-21 competent cell 2. Induction: separate 5ml out of 20ml overnight BL-21 as uninduced BL-21 stock and put into -80 degree, for the rest of 15ml, add 150ul 20% arabinose for induction, then culture overnight in 37 degree shaker |
| Results | None |

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September 11, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee |
| Experiments | Preparation for NSP4 activity assay  Colony picking |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1. Picked a colony from the transformation yesterday. Prepared a 20 mL culture with amp+ and incubated in 37 C at 6:49 am 2. Centrifuge the overnight induced culture of NSP4 Colony #3 BL-21, separate pellet and supernatant, then store in -80degree. Centrifuged 5mL uninduced sample NSP4\_plasmid#3 then stored in -80C. Induced the rest 15 mL (OD=1.1227 13-14 hr culture) with 150 ul 20% arabinose at 20:08 and incubated in shaking incubator. |
| Results | None |

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September 12, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee |
| Experiments | NSP4 activity assay |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | 1. Nickel pull down both induced and uninduced Nsp4 SUPERNATANT (new and old stock), total 4 samples 2. Scrapped OPHT2 bacteria (three stocks) from glycerol stock 082719 and cultured in ~5mL volume of Lb broth with amp+ and then put in 37C shaking incubator at 16:45. Glycerol stock for all OPHT2 colonies were thawed completely by accident watch out for the quality of this stock for next scrapping. 3. Homogenize 4 samples with tyrosinase assay buffer and test protein concentration with BCA protein assay (30mins incubation) |
| Results | None |

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September 13, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | OPHT2 induction  NSP4 activity assay  Material stock preparation |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | 1. Induced 5mL cultures of pet11a\_OPHT2 from yesterday with 1mM IPTG (10 ul of 500mM IPTG stock) and incubated at 37 C shaking incubator at 4:45. OD600 =0.7144 2. Tyrosinase activity assay started plate OD monitoring (510nm) at 8:02 am in the plate reader in 37 C temperature for 1 hr. Stored all proteins back in the “Protein sample box” in -80C 3. Made two 500mL LB broth and one 110 mL LB agar for making 10 amp+ plates. Made a stock of 50 mL LB Broth amp+ and put in 4 degree fridge. 4. Diluted induced pet11a\_OPHT2 #1 from early this morning. 1:1000 dilution ratio by putting 10 ul of that induced culture into 10 mL LB Broth amp+ then stored in shaking incubator 37 C overnight. |
| Results | Result of tyrosinase activity test today ![](https://static.igem.org/mediawiki/2019/7/73/T--UM_Macau--Lablog_13_Sep.jpg) |

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September 15, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | BL-21 OPHT2 #1 EGFP expression |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | eGFP expression is still very high even after ~28 hrs after induction for our bacteria Bl-21 OPHT2 colony #1 |
| Results | ![](https://static.igem.org/mediawiki/2019/7/7f/T--UM_Macau--Lablog_15_Sep.jpg) |

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September 16, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Material stock preparation  BL-21 OPHT2 #1 EGFP expression  Adhesion system reconstruction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1. Made 11 Amp+ agar plates by adding 100ul (1:1000) to 100ml agar solution. 2. eGFP expression is still very high even after ~40 hrs after induction for our bacteria Bl-21 OPHT2 colony #1 3. PCR reaction for the amplification of adhesion system for ligation to pet11a for 1 hr and 26 mins. Started at 15:34 |
| Results | ![](https://static.igem.org/mediawiki/2019/6/6f/T--UM_Macau--Lablog_16_Sep.jpg) |

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September 17, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee, Jia Ying |
| Experiments | Adhesion system reconstruction  Preparation digested pet11a vector stock |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | 1. Incubated a new BL\_21 OPHT2 colony #1, 5 mL volume, at 37 C  2. Run the gel with opht1,2,3 and opha1 samples (each well 30ul, each sample 2 wells) at 120V for 20 mins. Then gel purification.  3. Gibson assembly to ligate Pet11a vector with Opht1,2,3 and Opha1 inserts. Calculation as below:   |  |  |  | | --- | --- | --- | | Plasmid | Concentration(ng/ul) | pMol | | pet11a vector | 54.12 | 0.015 | | OPHT1 | 6.7 | 0.0068 | | OPHT2 | 7.4 | 0.0051 | | OPHT3 | 4.5 | 0.0045 | | OPHA1 | 17.3 | 0.018 |   75ng of vector=75/54.12=1.39ul  1.39ulx0.015pmol=0.02pmol  vector:insert=1:3  Insert=0.06pmol   |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | | Insert | Volume of insert(ul) | Volume of vector(ul) | Volume of water(ul) | Volume of Master Mix(ul) | Total volume  (ul) | | OPHT1 | 8.82 | 1.39 | - | 10 | 20.21 | | OPHT2 | 11.76 | 1.39 | - | 13 | 26.15 | | OPHT3 | 13.33 | 1.39 | - | 14 | 28.72 | | OPHA1 | 3.33 | 1.39 | 5.28 | 10 | 20 |   4.Restriction digestion for pet11a vector. We did it for 100ul, incubate for 14hrs at 37 degree, infinite hold at 4 degree. Calculation as below:  pET11a concentration: 49.1ng/ul   |  |  | | --- | --- | | pET11a (3ug) | 60ul | | BamHI | 2ul | | NHeI | 2ul | | Cutsmart buffer | 10ul | | ddH2O | 26ul | | Total | 100ul |   5. Transformation stock OPHT1, OPHT2, OPHT3, OPHM, and OPHA using BL-21; stock pBBR1MCS2\_OPHT2 using BL-21; stock pet11a\_OPHT1, OPHT2 using Rosetta. Put into the incubator at 21:00 |
| Results | Transformation:  ![](https://static.igem.org/mediawiki/2019/c/c4/T--UM_Macau--Lablog_17_Sep_1.jpg)  ![](https://static.igem.org/mediawiki/2019/d/de/T--UM_Macau--Lablog_17_Sep_2.jpg)  ![](https://static.igem.org/mediawiki/2019/2/20/T--UM_Macau--Lablog_17_Sep_3.jpg)  ![](https://static.igem.org/mediawiki/2019/8/8f/T--UM_Macau--Lablog_17_Sep_4.jpg)  ![](https://static.igem.org/mediawiki/2019/a/aa/T--UM_Macau--Lablog_17_Sep_5.jpg)  ![](https://static.igem.org/mediawiki/2019/c/ce/T--UM_Macau--Lablog_17_Sep_6.jpg)  ![](https://static.igem.org/mediawiki/2019/2/27/T--UM_Macau--Lablog_17_Sep_7.jpg)  ![](https://static.igem.org/mediawiki/2019/d/d9/T--UM_Macau--Lablog_17_Sep_8.jpg)  ![](https://static.igem.org/mediawiki/2019/f/fe/T--UM_Macau--Lablog_17_Sep_9.jpg)  ![]() |

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September 18, 2019
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|  |  |
| --- | --- |
| Performers | Julien, Renee, Jia Ying |
| Experiments | Colony picking for transformed bacteria  Bacteria transformation  Ammonia iron (III) citrate solution preparation  Induction of IPTG and Ammonia iron (III) citrate in OPHT2 bacteria  Gel run and gel purification |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Colony picking: Picked 1 colony for OPHT1,2,3,M and OPHA1 (BL-21), OPHT1,2 (Rosetta). Picked 3 colonies for pBBR1-Opht2 (BL-21). Incubate the picked colonies with 5ml Amp+ (for pET11a plasmid) or Kan+ (for pBBR1MCS2 plasmid) LB broth, put in 37degree shaking incubator at 4.30pm.   Transformation with new constructed pet11a\_OPHT, OPHT2, OPHT3 and OPHA using the Stbl3 strain E.coli. Put them into 37 degree incubator at 18:40.   Prepared the Ammonia iron (III) citrate solution at concentration:100mM with 26.3g iron(III) powder in 263mL water at 18:00.   We added 10ul IPTG and 1ml ammonium iron(iii) citrate into BL-21 OPHT2 at 6pm. We run the gel for digested pET11a vector, the expected band size is 5.6kb. Then we purify the excised gel (band1 weight 0.371g and band2 weight 0.372g) with 30ul elution buffer type 4 three times, each time incubated 5minutes. |
| Results | Gel run results: ![](https://static.igem.org/mediawiki/2019/6/6e/T--UM_Macau--Lablog_18_Sep.jpg)   The concentration for band1 is 2.24ng/ul and band 2 is 2.49ng/ul. |

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September 19, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Glycerol stock making  Colony picking and bacteria overnight culture |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Picked up adhesion system plasmids transformed plates from 37C, parafilmed and put it in 4C. All constructs have colonies. Picked three colonies for each transformed plasmids in Stbl3 with 5ml LB, incubated in 37 shaking incubator at 6.50pm.   Collected the overnight culture of picked colonies at 1.30pm [amplified adhesion system-old stock: OPHT1,2,3,M and OPHA1 (BL-21), OPHT1,2 (Rosetta) and pBBR1-Opht2 #1,2,3 (BL-21)]. Made 1ml glycerol stock of these with 500ul 50% glycerol and 500ul bacteria culture, stored in -80 degree fridge’s Glycerol stock box. |
| Results | Checked EGFP expression of pbbr1-opht2 with fluorescence microscope:  ![](https://static.igem.org/mediawiki/2019/2/2a/T--UM_Macau--Lablog_19_Sep_1.jpg)  ![](https://static.igem.org/mediawiki/2019/0/03/T--UM_Macau--Lablog_19_Sep_2.jpg)  ![](https://static.igem.org/mediawiki/2019/5/5b/T--UM_Macau--Lablog_19_Sep_3.jpg)  ![](https://static.igem.org/mediawiki/2019/6/6c/T--UM_Macau--Lablog_19_Sep_4.jpg)  ![](https://static.igem.org/mediawiki/2019/1/1b/T--UM_Macau--Lablog_19_Sep_5.jpg)  ![]() |

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September 20, 2019
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|  |  |
| --- | --- |
| Performers | Julien |
| Experiments | Sequencing samples preparation  Glycerol stock making |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Collected overnight culture of picked colonies (pet11a-stbl3) and prepared sequencing samples(1ml bacteria, 120ul 106FOR, 30ul 102 FOR), sent for sequencing.   Made 1ml glycerol stock with 500ul 50% glycerol stock and 500ul bacteria culture, stored in -80 degree fridge’s glycerol stock box. |
| Results | None |

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September 21, 2019
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|  |  |
| --- | --- |
| Performers | Julien |
| Experiments | Protein extraction |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Protein extraction for overnight bacteria culture (pet11a adhesion system-BL21, Rosetta) with 500ul lysis buffer, boiled at 98degree for 10minutes. Measured protein concentration after 30minutes BCA incubation. Run western blot at 1.40pm.   The order and volume loaded shown as below:  Ladder - OPHT1 (Rosetta) - OPHT2(Rosetta) - OPHT1 (BL-21) - OPHT2 (BL-21) - OPHT3 - OPHM - OPHA1  (5ul for ladder, 20ul for the rest protein samples)   Incubated with antibody and shaking in 4 degrees fridge at 5.20pm |
| Results | None |

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September 22, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Continue western blot |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Washed the membrane with about 3 times.   Incubated 10 mL OPHT2 Colony #1 in LB amp+ at 37 C shaking incubator at 23:30.  Magnetization of E.coli incubated in Ferric ammonium citrate(3 days incubation at 37 C) for 3 hrs with magnet. Made a video from the fluorescence microscope |
| Results | Western Blot results: ![](https://static.igem.org/mediawiki/2019/1/1c/T--UM_Macau--Lablog_22_Sep.jpg) |

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September 23, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Incubation of bacteria culture, adhesion system functional test |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Incubated 15 mL culture of all the new pET11a adhesion system glycerol stock at 37 C shaking incubator at 14:01. Will extract after 12 hrs.   Induced the overnight BL-21 with 1mM IPTG (50ul in 10 mL) and put in 37C shaking incubator. Inducing for 3 hrs.   Added 2 ml of 100mM ferric ammonium citrate into the 3 hr IPTG induced BL-21 OPHT2 colony #1(Checked egfp signal and it’s positive) and put it in 37C shaking incubator at 17:08. Incubating for 24 hrs.   Made 5 mL overnight culture of BL-21 for adhesion system functional test. Stored in 37 C shaking incubator at 20:50 . Used all colony #1 from the old pet11a glycerol stock. Incubating for 12 hrs. |
| Results | None |

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September 24, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Plasmid extraction  Induction of adhesion system  Magnetization test set up |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Extracted plasmids of the 15 mL new pet11a adhesion system and sent samples for sequencing.   Stored new pet11a plasmids in -20C in the new construct box.   Induced 5 mL overnight for functional test of adhesion system using 1mM IPTG(10 ul 500 mM stock -> 5 mL culture) for 3 hrs in 30C shaking incubator in Prof.Xu’s lab started at 16:30.   Started the magnetization test setups at 00:05. Prepared three dishes. Two that was incubated with fe3+ and one negative ctrl. One of the two dishes has been put in the fluorescence microscope and picture is being taken at 30 sec intervals for 6 hrs. |
| Results | None |

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September 25, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Incubation of bacteria culture |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Started the incubation of overnight culture of Tyrosinase (500 U/ml) 2 mL + 2 mL of our adhesion system bacteria that was resuspended with 10 uM Phosphate buffer (Which prepared at 092419). Incubated in Professor Xu's lab's shaking incubator temperature 25-27 C, shaking at 75 rpm.   Picked up our overnight culture E.coli with Tyrosinase at 19:45 (Total 17 hrs incubation).   Started the incubation of our "Activated E.coli with Tyrosinase" at 20:30. Mixed 150 ul of the nanoparticle PFODTBT (30 ppm) with the 4 mL overnight culture. Put the solution in Professor Xu's shaking incubator at 25C 70 rpm for 1 hr.   Put the activated bacteria incubated with the nanoparticle in the 4 Degree of PRofessor Xu's lab. |
| Results | ![](https://static.igem.org/mediawiki/2019/8/8b/T--UM_Macau--Lablog_25_Sep.jpg) |

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September 26, 2019
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|  |  |
| --- | --- |
| Performers | Julien |
| Experiments | Checking of nanoparticle binding with bacteria |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Centrifuged the nanoparticles incubated bacteria culture and resuspended the pellet with 1mL phosphate buffer. Get 10ul on glass slide and observed under a fluorescence microscope. We have negative ctrl(without tyrosinase), nanoparticles only and 5 bacteria samples. |
| Results | None |

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September 27, 2019
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|  |  |
| --- | --- |
| Performers | Julien |
| Experiments | Bacteria culture  Restriction digestion |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Prepare 5mL of pet11a adhesion system BL-21 bacteria culture with Amp+, shaking in 37 incubator of Prof. Henry’s lab.   Restriction digestion for pBAD4 vector and pBAD4-tyrosine vector with XbaI enzyme. Put in thermocycler 37 degree for 14 hours and 4 degree for infinite hold. |
| Results | None |

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September 28, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee |
| Experiments | DNA purification  Induction of samples  Gibson assembly  Transformation |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Separated 1ml culture as uninduced sample for each. Induced with 10uL IPTG for each samples except negative ctrl and incubated in Prof’s Xu lab 30 degree shaking incubator at around 11.40pm.   Purified DNA of digested pBAD24 and pBAD24\_NSP4 from agarose gels. Concentration of pBAD24 is 125.1ng/ul and pBAD24\_NSP4 is 61.31ng/ul.   Resuspend ftnA fragment with 25ul TE buffer followed by 50degree incubation for 20min. Gibson assembly of ftnA+pBAD24 and ftnA+pBAD24\_NSP4.   Gibson assembly for pBAD vector+ftnA and pBAD NSP4 +ftnA. Put in thermocycler for 1hr at 50degree. Calculation as below:  pBAD NSP4 conc=61.31ng/ul = 0.017pmol  pBAD conc=125.1ng/ul = 0.042pmol  ftnA conc=20ng/ul = 0.052pmol   100ng vector (pBAD NSP4)=1.63ul  (vector)1.63 x 0.017=0.028pmol  vector:insert=1:3  (insert)=0.083pmol  ftnA volume=0.083/0.052=1.6ul   |  |  | | --- | --- | | Vector | 1.63ul | | Insert | 1.6ul | | Master mix | 10ul | | water | 6.77ul | | total | 20ul |   100ng vector(pBAD)=0.8ul  (vector)0.8 x 0.042=0.034pmol  vector:insert=1:3  (insert)=0.102pmol  ftnA volume=0.102/0.052=1.96ul     |  |  | | --- | --- | | Vector | 0.8ul | | Insert | 1.96ul | | Master mix | 10ul | | water | 7.29ul | | total | 20ul |     Incubated our 1 mL bacteria with tyrosinase 500U/mL in Professor Xu’s shaking incubator at 25 C 90 rpm for 24 hrs.    Transformation of ftnA+pBAD24 and ftnA+pBAD24\_NSP4 into stbl3 strain. Start incubation at 22:17. |
| Results | None |

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September 29, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Colony picking |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Picked three colonies for FtnA+pBAD24 and made 25 mL bacteria solution with amp+ at 17:30. Unfortunately there were no colonies for the pBAD24\_NSP4+FtnA. Will repeat this transformation later. |
| Results | None |

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September 30, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Plasmid extraction |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Plasmid extraction for three overnight culture of pbad24-ftna. Used 30ul EB, repeated 3times and each time incubated 5 mins. Extracted concentration listed in "Results"   Incubated 100 ul of activated adhesion system + bacteria from 092819 with 10 ul (1 mg/mL concentration) of 1.0%w/v FP-00552-2 Yellow fluoresscent particle, 0.04-0.09 um size in 25C and mixing at 300 rpm for 1 hr. Started at 23:40 (Samples were covered with aluminum foil to prevent exposure to light). |
| Results | Concentration of extracted plasmid:  pBAD24-ftnA #1: 169.3ng/ul  pBAD24-ftnA #2: 200.9ng/ul  pBAD24-ftnA #3: 137.8ng/ul ![](https://static.igem.org/mediawiki/2019/1/10/T--UM_Macau--Lablog_30_Sep.jpg) |

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October 1, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Adhesion functon test |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Adhesion test fluorescence checking using 4th floor plate reader machine. Result: Failed will elaborate later. ![](https://static.igem.org/mediawiki/2019/5/5c/T--UM_Macau--Lablog_1_Oct.jpg)   Third functional test using 092819 1000ul activated bacteria. Put 5 ul of the yellow nanoparticle from Prof Xuanjun 30ppm into the 1000ul bacteria. Incubated at 25C for 1hr with shaking for 400 rpm. Started at 19:00.   Incubated new adhesion system bacteria for functional test. Picked all Colony #1. Prepared 5 mL overnight culture amp+, will subculture tomorrow to 20 mL culture (Need more bacteria concentration for the tests). Started the incubation at 22:30 at shaking incubator 37 C. |
| Results | None |

Close

October 2, 2019
×

|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Incubation of bacteria culture  Induction of adhesion system |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Subcultured 5 mL overnight cultures into 20 mL LB amp+ and incubated at 37 C for 3 hrs. Started incubation at 13:00. Saved the 5 mL overnight culture as uninduced samples. Will measure OD of both later, need OD=2 for activation and functional tests.   Induced Subcultured 20 mL adhesion system bacteria culture with 1 mM IPTG each (OPHT, OPHT2, OPHT3, OPHM, & OPHA). Incubated at 30 C for 4 hrs in Professor Xu's shaking incubator.   Update on the magnetization effect after incubation with ammonium ferric citrate of the BL-21 strain of E.coli. They can still be attracted to magnet until now since 092419.   Update on adhesion 20 mL culture, I will induce it overnight instead of 4 hrs. Will harvest them tomorrow morning instead. |
| Results | None |

Close

October 3, 2019
×

|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Checked sequences of newly constructed plasmid |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Checked the sequences of the newly constructed pet11a adhesion system plasmids. Conclusions are as follows：    OPHT1 colony # 3 = Should be correct (Has a single point mutation but it's peak looks like a sequencing error)  OPHT2 colony #1 = Correct and usable if needed  OPHT3 = no correct or intact colony  OPHA Colony #1 and #3 = Correct and usable if needed |
| Results | None |

Close

October 4, 2019
×

|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Sent sample for sequencing |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) |
| Records | Send ftna samples that Julien extracted from 093019 to Tech dragon sequencing. |
| Results | None |

Close

October 5, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Renee |
| Experiments | Functional test of the combination of tyrosinase and adhesion system |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Incubated 100ul bacteria with 50ul 1500U/ml tyrosinase and 1ul of 1%w/v yellow nanoparticle together for 4 hrs and 8hrs. Will check the fluorescence reading after these two time periods.    Cultured 20 mL bacteria using glycerol stock of colony number 1 for OPHT, OPHT2,OPHT3, OPHM, OPHA1 with Amp+ and one for BL21 only without Amp.    Current conclusions for adhesion system assay parameters: bacteria amount (Need OD=2), time of incubation with tyrosinase (4hrs, is the best I think), amount of tyrosinase (U/mL) (500U/ml or less), amount of nanoparticle (10ml/L or 10ppm is good for our experiment), centrifugation speed(3000xg 3 mins no nanoparticle pelleting), learning the plate reader machine(440 nm first filter and 480nm second filter), choosing the nanoparticle(1% w/v Yellow fluorescent particle from spherotech and red nanoparticle of Prof Xuan Jun), assay format in 96 well plate (As shown in the picture just now), Amount of bacteria and nanoparticle total volume (150 ul) so we can plate in 96 well.    ligated pBAD24\_NSP4 with fntA, and a negative control only has pBAD\_NSP4 without insert then transformed them into DH5 alpha. Started incubation at 20:23.    Cultured 18 tubes 20ml of bacteria from glycerol stock NSP4-Tyrosinase colony 3. |
| Results | Current conclusion based on this fluorescence plate reader result is that after 4 hrs of incubation with tyrosinase there seems to be a significant decrease of supernatant fluorescence signal compared to our inactivated supernatant samples. Most notable OPHM. ![](https://static.igem.org/mediawiki/2019/0/00/T--UM_Macau--Lablog_5_Oct.jpg) |

Close

October 6, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Renee |
| Experiments | Induction of NSP4 tyrosinanse |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Induced with 5 concentrations of arabinose in 15 tubes of nsp4 tyrosinase bacteria overnight culture. Another 3 tubes are not induced with arabinose act as negative ctrl. The volume of arabinose added shown as below: ![](https://static.igem.org/mediawiki/2019/a/a4/T--UM_Macau--Lablog_6_Oct_1.jpg)   Induced overnight OPHT1, OPHT2, OPHT3, OPHM and OPHA with 1 mM 40 ul of (500mM IPTG) per 20 mL culture and incubated in Professor Xu's shaking incubator 30 C for 4 hrs. Started ~approximately 12:08.    Collected 4 hr incubation with 5 concentrations arabinose’s bacteria culture.    Nickel pull down for nsp4 4 hr arabinose induced (supernatant only), stored the pulled down protein in -80c protein samples box.    Collected 8hr samples and centrifuging at 3000xg for 10 mins. Will separate supernatant into another tube.    Incubated adhesion 100219 and 100519 bacteria with yellow nanoparticle started incubation at approx~ 21:00.    Checked the construction of three extracted plasmids pbad24-fntA on 093019 by PCR and gel running.    Nickel pull down for nsp4 8 hr arabinose induced (supernatant only), stored the pulled down protein in -80 protein samples box.    Collected 12hr samples and centrifuging at 3000xg for 10 mins. Stored separated-supernatant into another tube and stored in -80c. |
| Results | Nanoparticle Fluorescent signal of inactivated and activated supernatant of OPHT, OPHT2, OPHT3, OPHM, OPHA, Negative control, and Positive control. ![](https://static.igem.org/mediawiki/2019/0/0c/T--UM_Macau--Lablog_6_Oct_2.jpg) |

Close

October 7, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Incubation of bacteria containing FtnA plasmid  Functional test of adhesion system  Tyrosinase activity assay |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Incubated transformed bl-21 with FtnA plasmids. Diluted outgrowth solution 1:100 and plated 350 ul of the bacteria. Put it in Prof Xu’s 37 degree incubator at 1:16 am.    Incubated BL-21 with pBAD24\_FtnA at 14:20 in Professor Xu's 37 C incubator. Only plated pBAD24\_FtnA#1 and pBAD24\_FtnA#2.    Tyrosinase activity assay for 4,8,12 hours incubation with arabinose at 5 different concentrations. We cultured them at 30 degree accidentally.  Tyrosinase activity assay reagents set-up:  Sample Background Control (SBC) mix: add this into SBC wells   |  |  | | --- | --- | | Assay buffer | 45ul x 18 = 810ul | | Enhancer | 5ul x 18 = 90ul |     Reaction mix: add this into assay background control, samples and positive control wells   |  |  | | --- | --- | | Assay buffer | 35ul x 21 = 735ul | | Substrate | 10ul x 21 = 210ul | | Enhancer | 5ul x 21 = 105ul |   Positive control well: 2ul positive control + 48ul assay buffer  Assay background control: 50ul Assay buffer  Plate: ![](https://static.igem.org/mediawiki/2019/0/06/T--UM_Macau--Lablog_7_Oct.jpg) |
| Results | Results for the adhesion system functional test. Conclusion: The results are quite consistent. Plate was read three times with the same machine and averaged all the reads as shown in the picture data set. Then I averaged each the three repeats for each samples and obtained final reads for each 100219 and 100519 samples and made the graph based on them. We can see a clear difference with the activated and inactivated one. Negative control (Wild type, just BL-21) is too close to our inactivated samples, not sure if the activated readings would be considered significant, but this negative control sample has been prepared along with the 100519 sample so it actually cannot act as a negative control for the 100219 samples but I just put it anyway. We need a third biological repeat for all these to have a final conclusion. Anyways, the data is very consistent, activated sample's readings are always lower than the inactivated ones for these first two biological repeats. ![](https://static.igem.org/mediawiki/2019/5/56/T--UM_Macau--Lablog_7_Oct_2.jpg) ![](https://static.igem.org/mediawiki/2019/3/36/T--UM_Macau--Lablog_7_Oct_3.jpg)   The results of plates after 1 hour measurement in the plate reader (30seconds interval, 37 degree incubation, OD 510nm) is saved. |

Close

October 9, 2019
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|  |  |
| --- | --- |
| Performers | Julien |
| Experiments | Nickel pull-down for NSP4 tyrosinase protein |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Incubated 15 tubes of 5ml nsp4 bacteria with 5 different arabinose concentration (2%, 0.2%, 0.02%, 0.002% and 0.0002%) at 12.30pm.     |  |  | | --- | --- | | Concentrations of arabinose | Volume to add | | 2% | 500ul | | 0.2% | 50ul | | 0.02% | 5ul | | 0.002% | 0.5ul | | 0.0002% | 0.05ul |     Collected nsp4 tyrosinase bacteria after 4hr incubation (at 4.30pm), centrifuged and keep the supernatant. Nickel pull down for 4hr incubation batch nsp4 bacteria’s protein and stored in -80 fridge’s bacteria box. (Because no space in protein sample box). Collected 8 hr incubation of different concentrations arabinose in nsp4 at 8.30pm. nickel pull down for 8hr incubation batch nsp4 bacteria’s protein and stored in -80 fridge’s bacteria box. (Cuz no space in protein sample box). Collected 12 hr arabinose incubation at 5 different concentrations of nsp4 bacteria at 12.30am . Centrifuged and kept the supernatant in -80 with a green holder. |
| Results | None |

Close

October 10, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Nickel pull down |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Nickel pull down   Diluted protein samples by 10x and measure the protein concentration using BCA assay. |
| Results | Protein concentration: ![](https://static.igem.org/mediawiki/2019/6/63/T--UM_Macau--Lablog_10_Oct.jpg) |

Close

October 11, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien, Pinto |
| Experiments | Tyrosinase activity assay  Protein extraction  Western Blot |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Tyrosinase activity assay for 4,8,12 hours incubation with arabinose at 5 different concentrations. We cultured them at 37 degree this time.  Tyrosinase activity assay reagents set-up:  Sample Background Control (SBC) mix: We use only one SBC for each time point incubation of protein samples as we were running out of reagents.   |  |  | | --- | --- | | Assay buffer | 45ul x 3 = 135ul | | Enhancer | 5ul x 3 = 15ul |     Reaction mix: add this into assay background control, samples and positive control wells   |  |  | | --- | --- | | Assay buffer | 35ul x 21 = 735ul | | Substrate | 10ul x 21 = 210ul | | Enhancer | 5ul x 21 = 105ul |     Positive control well: 2ul positive control + 48ul assay buffer  Assay background control: 50ul Assay buffer    Plates: ![](https://static.igem.org/mediawiki/2019/5/5c/T--UM_Macau--Lablog_11_Oct.jpg) SDS-page gel loading sequence:  First gel (OPHT-adhesion system):  Ladder- Ladder- Control- OPHT1 - OPHT2 - OPHT3 - OPHM - OPHA1    Second gel (ftNA):  Ladder - Control - ftnA1.1 - ftnA1.2 - ftnA1.3 - ftnA2.1 - ftnA2.2 - ftnA2.3 |
| Results | In the functional test results page |

Close

October 12, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Renee |
| Experiments | ftnA functional test |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Centrifuged the second bench ftnA test samples and resuspend in LB broth. Measure the OD value. Begin magnet attraction test at 9:30 and will check their states every hour.  ![](https://static.igem.org/mediawiki/2019/d/d4/T--UM_Macau--Lablog_12_Oct_1.jpg)  ![](https://static.igem.org/mediawiki/2019/1/12/T--UM_Macau--Lablog_12_Oct_2.jpg)  ![](https://static.igem.org/mediawiki/2019/e/ec/T--UM_Macau--Lablog_12_Oct_3.jpg)   Transformation of pBAD24-FtnA #2 into seven strains which are Stbl3, DH5 alpha, BL-21 DE3, BL-21 Star, BL-21 Rosetta, BL-21 pLys, TOP10. Started incubation at 13:00.   Reset pBAD24-FtnA 1.1, 1.2, 2.1, 2.2 attraction test with only one magnet under it at 14:50.   Induced 5 tubes 5ml bacteria pBAD24-FtnA 1.1 with 1ml 100mM ferric ammonium citrate at 16:20.   101219: Incubated 20 mL BL-21 NSP4 tyrosinase on 37 degree for adhesion system functional test with our own enzyme. |
| Results | None |

Close

October 13, 2019
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|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Adhesion system functional test |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Started adhesion system functional test. Incubated adhesion system bacteria with yellow nanoparticle. Would collect the samples at 4 different time points, 1 hr, 2 hr, 3 hr and 4 hr and compare the difference in the relative fluorescence. |
| Results | None |

Close

October 14, 2019
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|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Bacteria culture, Incubate with antibody for immunoblotting membrane |
| Protocols | [Plasmid Construction](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf) [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Incubated 5ml culture of FtnA-Stbl3,Dh5alpha,BL-21(De3),BL-21(star), BL-21(plys), TOP10, BL-21(Rosetta), And Bl21(WT) at 37 degree shaking incubator started 14:39.   Pinto made SDS gel and store in Buffer. Measured protein concentration of FtnA protein samples and stored in -80C.   Autoclaved some tips blue and yellow tips and 1.5 mL centrifuge tubes.   Incubated FtnA samples and RpoB internal control with anti-flag and anti-rabbit primary antibody. Put 5 mL and 3 mL respectively of the antibodies. Put in 4 degree in Henry’s fridge at 20:20.   Incubated 6 20mL culture of OPHT, OPHT2,OPHT3, OPHM, OPHA and WT bl-21 all picked from glycerol stock colony #1 in 37 degree shaking incubator overnight(start 21:00) Has white tape mark, will induce it with 1mM IPTG for 4 hrs after 12 hrs (9:00). For time dependent functional test with yellow fluorescent nanoparticle and Professor Xuan Jun’s nanoparticle.   Incubated 5x 20 mL culture of NSP4\_BL21 subcultured from yesterday’s overnight culture(1:100 ratio) at 37C started at (21:00) . Will induce later with 0.2% arabinose after 3hrs (00:00). And let it produce tyrosinase into the supernatant for overnight(12-16 hrs) Has green tape mark. This is for testing our own secreted tyrosinase whether it would be able to activate our capture system bacteria.   Incubated 6x 5mL tubes of OPHT,OPHT2,OPHT3,OPHM, OPHA and negative ctrl at 37 C has yellow tape mark started (21:00). Picked from glycerol stock colony #1. Will incubate overnight and induce tomorrow after 16 hrs with IPTG and incubate in 30C for 4 hrs (12:00).   Incubated 7x 5mL tubes for each bacteria strains we had cultured overnight FtnA-Stbl3,Dh5alpha,BL-21(De3),BL-21(star), BL-21(plys), TOP10, BL-21(Rosetta), And Bl21(WT) at 37 degree shaking incubator started 21:00. Has red tape. Subcultured 1:100 ratio will induce with 0.2% arabinose later after 3 hrs (00:00) and incubate for 12-16 hrs. These samples are for protein concentration check for FtnA based on different strains. Will extract proteins tomorrow and measure protein concentration along with the preparation of protein samples for western blot running on Wednesday.   Induced earlier log picked strains incubated this morning from 2:39 5ml cultures of FtnA-Stbl3,Dh5alpha,BL-21(De3),BL-21(star), BL-21(plys), TOP10, BL-21(Rosetta), And Bl21(WT) with 0.2% arabinose (50 ul of 20% arabinose) and stored in 37 degree shaking incubator has purple tape. Started at 21:42. Will incubate overnight for 12 hrs. Then would put with ~20 mM ammonium ferric Citrate 1 mL each and incubate for 24 hrs for magnetization test on Wednesday morning 9:00 am. |
| Results | None |

Close

October 15, 2019
×

|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Western blot |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Induced red and green taped samples from previous logs with 0.2% arabinose at 00:44 and put in the 37 C shaking incubator. Plan to incubate for 12-16 hrs (Probably 16 hrs).   Induced yellow tape marked tubes and white marked tape tubes with 1mM IPTG at 12:00, put them in 30C shaking incubator of Professor Xu's lab.   Washed the membrane with tbst 3 times and incubated with anti-rabbit 2nd antibody at 12.40pm |
| Results | None |

Close

October 16, 2019
×

|  |  |
| --- | --- |
| Performers | Philip, Julien |
| Experiments | Western blot |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Incubated purple taped samples from 101419 with ~20 mM ferric ammonium citrate. At 37 C starting 00:49.  Run SDS-Page gel. Loading order as below:  First gel  Ladder-Ladder-Control-2%-0.2%-0.02%-0.002%-0.0002%    Second gel  Ladder-BL-21WT-Rosetta-BL-21(star)-TOP10-BL-21(plys)-Dh5alpha-BL-21(De3)    Incubated membranes with primary antibody for overnight. (Anti-flag for 19kda ftna, Anti-rpob for 150kda internal ctrl). |
| Results | None |

Close

October 17, 2019
×

|  |  |
| --- | --- |
| Performers | Philip |
| Experiments | Immunofluorescence |
| Protocols | [Protein Expression](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf) |
| Records | Incubated immunofluorescence slide samples with histidine primary antibody for 12-16 hrs. Put in 4 degree in Henry’s fridge. |
| Results | None |

Close

October 18, 2019
×

|  |  |
| --- | --- |
| Performers | Philip, Renee |
| Experiments | Cell viability testing |
| Protocols | [Functional Test](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf) |
| Records | Started incubation of bacteria cell viability with ZnO and AgNP at 04:30. Will test the following time points (Hrs) or OD600 (2, 4,6,8,10,12,14,16). 6:30, 8:30, 10:30, 12:30, 14:30, 16:30, 18:30 are the time points. I would need help getting the absorbance values for the 10:30, 12:30, 14:30 timepoints.   Incubated 20 mL overnight culture OPHt,OpHT2, OPHT3, OPHM, OPHA, -ctrl in 37 C shaking incubator started 17:00. |
| Results | None |

Close

[![](https://static.igem.org/mediawiki/2019/6/63/T--UM_Macau--documenticon.png)](https://static.igem.org/mediawiki/2019/c/c0/T--UM_Macau--Plasmid_Construction.pdf)

### Plasmid Construction

[![](https://static.igem.org/mediawiki/2019/6/63/T--UM_Macau--documenticon.png)](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--Protein_Expression.pdf)

### Protein Expression

[![](https://static.igem.org/mediawiki/2019/6/63/T--UM_Macau--documenticon.png)](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf)

### Functional Test

![](https://static.igem.org/mediawiki/2019/9/96/T--UM_Macau--homepicigem.png)

#### CONTACT US

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---

###### iGEM 2019 UM\_Macau


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:UM Macau/Experiments

[UM\_Macau](https://2019.igem.org/Team%3AUM_Macau)

* Team

  [Team Members](https://2019.igem.org/Team%3AUM_Macau/Team)
  [Attribution](https://2019.igem.org/Team%3AUM_Macau/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AUM_Macau/Description)
  [Experiment Design](https://2019.igem.org/Team%3AUM_Macau/Experiments)
  [Results](https://2019.igem.org/Team%3AUM_Macau/Results)
  [Notebook](https://2019.igem.org/Team%3AUM_Macau/Notebook)
  [Modeling](https://2019.igem.org/Team%3AUM_Macau/Model)
  [Demonstrate](https://2019.igem.org/Team%3AUM_Macau/Demonstrate)
* Parts

  [Basic Part](https://2019.igem.org/Team%3AUM_Macau/Basic_Part)
  [Composite Part](https://2019.igem.org/Team%3AUM_Macau/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3AUM_Macau/Part_Collection)
* [Lab Safety](https://2019.igem.org/Team%3AUM_Macau/Safety)
* Human Practice

  [Integrated Human Practice](https://2019.igem.org/Team%3AUM_Macau/Human_Practices)
  [Public Engagement](https://2019.igem.org/Team%3AUM_Macau/Public_Engagement)
  [Collaboration](https://2019.igem.org/Team%3AUM_Macau/Collaborations)
* Achievement

  [Judging Form](https://igem.org/2019_Judging_Form?team=UM_Macau)
  [Medal Criteria](https://2019.igem.org/Template%3AUM_Macau/Medal_Criteria)

# Experiment Design

[![](https://static.igem.org/mediawiki/2019/3/36/T--UM_Macau--homepicdownarrow.png)](#pagestart)

# Design Overview

Our project aims to engineer an *Escherichia coli* that is capable to capture nanoparticles, activate itself and be able to be controlled through magnetization. Our efforts are to demonstrate the proof of concept that a controllable cleaner microorganism is possible. In the future, teams can use our results as a basis for a controllable wastewater cleaning organism that is safe and cheaper to mass produce for the application of helping improve water cleaning efficiency.

This humble project demonstrates the first step into developing a wastewater cleaning organism. A proof of concept, our engineered) *E.coli* uses a sticky protein on the cell surface called L-Dopa; which would be derived from tyrosine, in order to capture nanoparticles as our first feature. As a second feature our organism would also be able to secrete a functional tyrosinase enzyme in order to convert tyrosine into L-Dopa, which is the primary domain wherein nanoparticles or other suspended particles could bind into. As a third feature, we would demonstrate that our *E.coli* is magnetizable through the overexpression of FtnA which could store iron ions and make our *E.coli* magnetizable. Hopefully with our organism able to capture nanoparticles we are able to reduce the nanoparticle concentration in the suspended solution during the clarifier stage of the wastewater treatment plants.

Our Self Activating Nanoparticle Collector *E.coli* would be constructed in three phases as shown in Figure 1. First we would construct the plasmid which would contain our different composite parts (BBa\_K3033018, BBa\_K3033019, BBa\_K3033020, BBa\_K3033021, BBa\_K3033022, BBa\_K3033023 and, BBa\_K3033024). We would primarily use the *E.coli* strain BL-21(DE3) which is a suitable *E.coli* strain for protein expression due to it being deficient of proteases Lon and OmpT which might affect our protein integrity. [7]

![](https://static.igem.org/mediawiki/2019/8/84/T--UM_Macau--ExDesign_fig1.png)

Figure 1. General workflow of the project.

1st Feature: Surface Nanoparticle Capturing Protein (SNCP)

![](https://static.igem.org/mediawiki/2019/1/1f/T--UM_Macau--ExDesign_1st_feature.png)

![](https://static.igem.org/mediawiki/2019/2/27/T--UM_Macau--ExDesign_fig2.png)

Figure 2. Overview structure of the surface capturing system.

Our surface nanoparticle capturing protein (SNCP) has a couple of components as shown in Figure 2. An anchoring protein. A linker enzyme. Next is the presence of 6-histidine tag. Finally, the capturing domain. Our inspiration for this capturing system is from the Mefp-5 protein that is found on the foot of the mussels. This inspiration protein has L-Dopa and histidine repeats in them thus we want to express L-Dopa as our capturing domain as it has a sticky property.

Our system would primarily be anchored onto the cell membrane of the bacteria using the outer membrane protein called OmpW derived from E. coli itself, it forms an 8-stranded beta-barrel with a long and narrow hydrophobic channel. We chose this as our anchoring protein as it is very stable and is able to carry our other protein onto the surface of the bacteria. We have truncated this version of OmpW at the 191st amino acid as it has been shown to be stably anchored onto the cell membrane while being able expose the rest of the proteins onto the outside of the cell membrane. [1]

![](https://static.igem.org/mediawiki/2019/4/49/T--UM_Macau--ExDesign_fig3.png)

Figure 3. Structure of OmpW [2]

The next component is the Perhydrolase which was derived from the organism *Pseudomonas aeruginosa*. According to our adviser, this would be enhancing our capturing system’s capability thorough the process of epoxidation. The next component is the 6- histidine tag which would be used to check the protein expression.

![](https://static.igem.org/mediawiki/2019/a/ac/T--UM_Macau--ExDesign_fig4.png)

Figure 4: Variants of our capturing system domains

Finally, we have the capturing domain wherein we have 4 different variation as shown in Figure 4. 2-Tyrosine, 6-Tyrosine, Mefp-5, and 2-Aspartic acid. We decided to modify the capturing domain to see whether which variant would be more effective at capturing nanoparticles. The 2-Tyr variant was chosen as it has been shown to be able to be converted to L-DOPA and bind to many surfaces, we used this variant as the baseline and modified accordingly.[1] We have added more tyrosine to see whether the addition of more tyrosine would further enhance the capturing function. Mefp-5 protein from Mussels itself was also used as a variant to compare the effectiveness of our other variants. 2-Aspartic acid was chosen due to a hypothesis the carboxyl group of found in the capturing domain is actually the main reason for the sticky function. In all of these variants except for Aspartic acid, would require the conversion of Tyrosine into L-Dopa using the tyrosinase which would be our second feature.

![](https://static.igem.org/mediawiki/2019/1/1f/T--UM_Macau--ExDesign_fig5.png)

Figure 5: Biobrick design (Variations of OPHT part)

The design of our constructs for the capturing system are as shown in figure 5. We used a T7 promoter (BBa\_K3033000) to drive the expression of our whole fusion protein. Then a Ribosome binding site (BBa\_K3033002) in order to translate our fusion protein. It is then followed by the anchoring protein (BBa\_K3033004) then the perhydrolase (BBa\_K3033005) as a linker an enhancer of our sticky function through epoxidation. A histidine tag (BBa\_K3033006) was put for later checking of protein expression using western blot. Our capturing domain which has 4 variants as shown earlier: OPHT with 2-tyrosine (BBa\_K3033007), OPHTIII with 6-tyrosine (BBa\_K3033008), OPHM with Mefp-5 (BBa\_K3033010), and OPHA 2-Asp (BBa\_K3033011). We also have made a variant of OPHT and put an eGFP tag on the N-terminal of the protein and thus we have made the OPHTII construct for an easy reporter checking the successful induction of *E.coli* and checking the localization of our protein. Finally, we have put a transcription terminator at the end which was derived from the pET11a plasmid (BBa\_K3033015) so that the bacteria would not be wasting energy. Our capture protein fragments would be ligated into the pet11a plasmid backbone.

2nd Feature: Tyrosinase Secretion and Activation System

![](https://static.igem.org/mediawiki/2019/b/b3/T--UM_Macau--ExDesign_2nd_feature.png)

Tyrosinase is an oxidase which we would be using to synthesize L-DOPA on the cell surface of our engineered organism. We are using this enzyme to convert our expressed L-tyrosine on the capturing domains of the surface capture protein to L-DOPA. The chemical is shown in Figure 6a below. We have used a tyrosinase coding sequence (BBa\_K3033013) which was derived from the tyrosinase producing bacteria called Bacillus megaterium strain M36. [3]

![](https://static.igem.org/mediawiki/2019/c/cf/T--UM_Macau--6aaa.png)

Figure 6. A: Chemical reaction where L-Tyrosine is converted to L-DOPA through the hydroxylation of the monophenol domain into o-diphenols. [6] B: The Sec-A dependent pathway for secretion of our protein of interest. [5]

Our second feature is a tyrosinase secretion system. This is a necessary system to have for the whole idea of our project due to the inability for L-Dopa to be synthesized naturally by *E.coli*. Therefore, the modified *E.coli* would be secreting an active form of tyrosinase into the supernatant where our organism would be located in, so that it would have contact with our capturing domains and convert tyrosine to L-Dopa. We plan to use the NSP4 signal sequence to carry our protein of interest to extracellular environment [4] Through the Signal Recognition Particle mediated sec-dependent pathway A as shown in Figure 6b above our protein would be secreted to outside of the cell. [5]

![](https://static.igem.org/mediawiki/2019/d/db/T--UM_Macau--ExDesign_fig7.png)

Figure 7. The composite part for our second feature, tyrosinase secretion system.

Our secretion system is designed as shown in, figure 7. We have constructed the secretion system to be under the araBAD promoter (BBa\_K3033001), we would use the ribosome binding site (BBa\_K3033003) to initiate the translation of the tyrosinase protein. 6 bp after the ribosome binding site we have put the secretion signaling peptide NSP4 (BBa\_K3033012) as a signaling tag for our coding sequence of the Tyrosinase enzyme which was derived from Bacillus megaterium (BBa\_K3033013). We then tagged our protein with 6-Histidine (BBa\_K3033006) for later protein expression check. A rrnB T1 Terminator (BBa\_K3033016) is derived from the pBAD24 plasmid to stop the transcription of unnecessary nucleotides to avoid taxing the cell’s machinery.

![](https://static.igem.org/mediawiki/2019/1/1c/T--UM_Macau--ExDesign_fig8.png)

Figure 8. Surface capturing and tyrosinase secretion and activation system.

As shown in Figure 8 above, the first step is to express the capturing proteins on outermembrane. The second step is to secrete the active form of tyrosinase, onto the supernatant wherein our capturing system is exposed, then it would react with the capturing domain to convert L-tyrosine into L-DOPA. After this second step, the capturing system’s capturing domain is now ready to bind onto our nanoparticle targets.

3rd Feature: Magnetization and Control System

![](https://static.igem.org/mediawiki/2019/0/0f/T--UM_Macau--ExDesign_3rd_feature.png)

The precise spatial arrangement of cells has been one of the most important topics in synthetic biology, and this feature also plays important role in our project, SANCE. After the surface capturing system is activated by self-secreting tyrosinase and capture nanoparticles by the adhesive fusion protein on its surface, the *E.coli* – nanoparticles complexes should be removed from the wastewater treatment system to complete its mission. Hence, we designed our *E.coli* to obtain the third feature of our project, the magnetization. With this feature, our *E.coli* could be magnetized and be removed away from wastewater treatment process when a magnetic field is applied as shown in Figure 9.

![](https://static.igem.org/mediawiki/2019/8/84/T--UM_Macau--ExDesign_fig9.png)

Figure 9: We aim to make our *E.coli* magnetizable so that we are able to collect them when they are applied to any waste water tank and extract them.

We accomplished this feature by cloning and overexpressing FtnA, which encodes for a globular protein complex called ferritin, consists of 24 protein subunits to form a nanoshell to store iron molecules by various metal-protein interactions ("Ferritin protein nanocages—the story"), Figure 10 shows the protein structure. Therefore, with the overexpression of FtnA, more ferritin proteins can help *E.coli* to store more Fe3+ ions within cytosol. [8] We expect that the magnetization of *E.coli* could be achieved within 30 minutes to 1 hour in real time observation. Thus, the removal of *E.coli* – nanoparticles complexes in wastewater treatment could be done within short duration.

![](https://static.igem.org/mediawiki/2019/2/2d/T--UM_Macau--ExDesign_fig10.png)

Figure 10: The protein structural feature of *E.coli* ferritin. [9]

Our team has overexpressed the FtnA using the composite part BBa\_K3033024 as shown in Figure 11 which would be ligated into a pBAD24 vector. The composite part is made up of the following components: (BBa\_K3033001) which is the pBAD promoter, (BBa\_K3033003) which is the ribosome binding site that is based on the pBAD24 plasmid, (BBa\_K3033014) is the FtnA coding sequence gene which is derived from the *E.coli* organism, (BBa\_K3033017) which is for the flag tag for later analysis of protein expression by doing western blotting, (BBa\_K3033016) is the pBAD24 derived terminator for stopping the transcription of unnecessary nucleotides.

![](https://static.igem.org/mediawiki/2019/6/64/T--UM_Macau--ExDesign_fig11.png)

Figure 11: Composite part for the overexpression of FtnA, the gene is under the promoter pBAD and the Arabad RBS.

# Reference

1. Park J. P., Choi M.J., Kim S. H., Lee S. H., Lee H. (2014). Preparation of Sticky Escherichia coli through surface display of an adhesive Catecholamine Moeity. Applied and Environmental Microbiology. 80(1) p.43-53.
2. Hong H., Patel D. R., Tamm L. K., Berg B. V. D. (2005) The Outer Membrane Protein OmpW forms an Eight-stranded beta-Barrel with a Hydrophobic Channel. The journal of biological chemistry. 281(11) pg. 7568-7577.
3. Arikan B., Valipour E., (2015). Increased production of Tyrosinase from Bacillus megaterium strain M36 by the response surface method.
4. Han S.J., Machhi S., Berge M., Xi G., Linke T., Schoner R. (2017). Novel signal peptides improve the secretion of recombinant staphylococcus aureus. Alpha toxinh35L in Escherichia coli.
5. Bechkwith J., (2013). The Sec-dependent pathway. Microbiology. 164 pg 497-504
6. Agarwal P., Singh M., Signh J., Singh R. P., (2019). Microbial Tyrosinases: A Novel Enzyme, Structural Features, and Applications. Applied Microbiology and Bioengineering. pg 3-19.
7. Muhlmann M., Buchs J., Noack S., Forsten E.(2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb Cell Fact. 16(1), pg 200.
8. Bitoun J. P., Wu G., Ding H., (2008). Escherichia coli FtnA acts as an Iron Buffer for Re-assembly of Iron- Sulfur Clusters in Response to hydrogen Peroxide Stress. Biometals. 21(6) 693-703.
9. Image from the RCSB PDB (rcsb.org) of PDB ID 1EUM (T.J. Stillman, P.D. Hempstead, P.J. Artymiuk, S.C. Andrews, A.J. Hudson, A. Treffry, J.R. Guest, P.M. Harrison) (2001) The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives. J.Mol.Biol. 307: 587-603.)

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###### iGEM 2019 UM\_Macau

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# Team:UM Macau/Results

[UM\_Macau](https://2019.igem.org/Team%3AUM_Macau)

* Team

  [Team Members](https://2019.igem.org/Team%3AUM_Macau/Team)
  [Attribution](https://2019.igem.org/Team%3AUM_Macau/Attributions)
* Project

  [Description](https://2019.igem.org/Team%3AUM_Macau/Description)
  [Experiment Design](https://2019.igem.org/Team%3AUM_Macau/Experiments)
  [Results](https://2019.igem.org/Team%3AUM_Macau/Results)
  [Notebook](https://2019.igem.org/Team%3AUM_Macau/Notebook)
  [Modeling](https://2019.igem.org/Team%3AUM_Macau/Model)
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  [Basic Part](https://2019.igem.org/Team%3AUM_Macau/Basic_Part)
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  [Part Collection](https://2019.igem.org/Team%3AUM_Macau/Part_Collection)
* [Lab Safety](https://2019.igem.org/Team%3AUM_Macau/Safety)
* Human Practice

  [Integrated Human Practice](https://2019.igem.org/Team%3AUM_Macau/Human_Practices)
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  [Judging Form](https://igem.org/2019_Judging_Form?team=UM_Macau)
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# Results

[![](https://static.igem.org/mediawiki/2019/3/36/T--UM_Macau--homepicdownarrow.png)](#pagestart)

1st Feature: Surface Nanoparticle Capturing Protein (SNCP)

# I. Plasmid Construction

## Pet11a-OPHT1/OPHT2/OPH3/OPHM/OPHA

### PCR and gel running results

![](https://static.igem.org/mediawiki/2019/f/fa/T--UM_Macau--a1.png)

**Figure A-1:** Gel result of the PCR amplification for clone identity checking for OPHT1 and OPHT2

We amplified OPHT1 DNA fragment from plasmids of colonies #1, #2, #3 and OPHT2 DNA fragment from plasmids of colonies #1, #2 by using PCR. The OPHT1 fragment bands appear slightly below 1500bp size and OPHT2 fragment bands are at around 2000bp; which indicated the pET11a-OPHT1 and pET11a-OPHT2 plasmid have been successfully constructed.

![](https://static.igem.org/mediawiki/2019/7/73/T--UM_Macau--a2.png)

**Figure A-2:** Gel result of the PCR amplification for the clone identity checking for OPHT3 and OPHM

We amplified OPHT3 DNA fragment of colonies #1, #2 and OPHTM DNA fragment of colonies #1, #2 by using PCR. The OPHT3 fragment bands appear slightly below 1500bp size and OPHTM fragment bands are at the range between 1500~2000bp; which indicated that pET11a-OPHT3 and pET11a-OPHTM plasmid have been successfully constructed.

![](https://static.igem.org/mediawiki/2019/8/80/T--UM_Macau--a3.png)

**Figure A-3:** Gel result which shows the amplified PCR product which pertains to OPHA.

We amplified OPHA DNA fragment from plasmids of colonies #1, #2 through PCR. The OPHA fragment bands appear slightly below 1500bp size; which indicated that pET11a- OPHA plasmid is successfully constructed.

### OPHT1 plasmid’s sequencing result

In all the plasmid sequencing results figures below, each white gap in between primer pairs of each colony indicates a mismatched pair.

![](https://static.igem.org/mediawiki/2019/c/ca/T--UM_Macau--a4.png)

**Figure A-4:** Sequencing result for the OPHT1 variant of the Surface nanoparticle capturing protein (SNCP).

From the plasmid sequencing results for OPHT1 colony 1,2 and 3, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony.

### OPHT2 plasmid’s sequencing result

![](https://static.igem.org/mediawiki/2019/1/1a/T--UM_Macau--a5.png)

**Figure A-5:** Sequencing result for the OPHT2 variant of the Surface nanoparticle capturing protein (SNCP).

From the plasmid sequencing results for OPHT2 colony 2 and 5, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony.

### OPHT3 plasmid’s sequencing result

![](https://static.igem.org/mediawiki/2019/a/ab/T--UM_Macau--a6.png)

**Figure A-6:** Sequencing result for the OPHT3 variant of the Surface nanoparticle capturing protein (SNCP).

From the plasmid sequencing results for OPHT3 colony 2 and 4, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony. On the other hand, there are a lot of mismatch pairs in colony 3, therefore the bacteria culture from this colony is not appropriate to be used.

### OPHM plasmid’s sequencing result

![](https://static.igem.org/mediawiki/2019/a/a3/T--UM_Macau--a7.png)

**Figure A-7:** Sequencing result for the OPHM variant of the Surface nanoparticle capturing protein (SNCP).

From the plasmid sequencing results for OPHM colony 2,3 and 4, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony.

### OPHA plasmid’s sequencing result

![](https://static.igem.org/mediawiki/2019/7/79/T--UM_Macau--a8.png)

**Figure A-8:** Sequencing result for the OPHA variant of the Surface nanoparticle capturing protein (SNCP).

From the plasmid sequencing results for OPHA colony 1 and 2, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony. There are more mismatch pairs in colony 3 compared to colony1 and 2.

# II. Protein Expression Check Results

### Western blotting results: Surface nanoparticle capturing protein variants

![](https://static.igem.org/mediawiki/2019/4/42/T--UM_Macau--a9.png)

**Figure A-9:** This western blotting result shows the band signals for all the variants for our surface nanoparticle capturing protein (SNCP)

### Immunofluorescence: Surface nanoparticle capturing protein variants

![](https://static.igem.org/mediawiki/2019/a/a3/T--UM_Macau--a10.png)

**Figure A-10:** Immunofluorescence imaging result of our bacteria transformed with our Surface nanoparticle capturing protein (SNCP)

We want to examine whether our target protein is being expressed in our 5 different surface NPs capturing protein: OPHT1, OPHT2, OPHT3, OPHM, and OPHA. From Figure A-9, we can observe that our OPHT1, OPHT2, and OPHA show the band on the correct size. OPHT1 and OPHA1 show bands at around 50kDa, while OPHT2 which tagged with GFP shows the band at around 75kDa; which are at the estimated protein band size. However, unfortunately, OPHT3 and OPHM did not show any protein band. Although there are no positive results from western blot in OPHT3 and OPHM, the green light from the anti-mouse secondary antibody can be seen in immunofluorescence (IF) assay which shows that the all adhesion constructive system express on the surface of our bacteria (Figure A-10).

# III. Functional Test

Yellow fluorescent nanospheres from Spherotech catalog number FP-00552-2 with and excitation wavelength of 440 nm and emission wavelength of 480 nm; with the size of 40 nm to 90 nm were used in all of the test. Refer to our protocol file for the details of the assay: [Click here!](https://static.igem.org/mediawiki/2019/b/b9/T--UM_Macau--Functional_Test.pdf)

![](https://static.igem.org/mediawiki/2019/3/3d/T--UM_Macau--a11_a12.png)

**Figure A-11:** RFU of 5 variants and wild-type bacteria after 6-minute treatment

**Figure A-12:** RFU of 5 variants and wild-type bacteria after 12-minute treatment

![](https://static.igem.org/mediawiki/2019/e/e0/T--UM_Macau--a13_a14.png)

**Figure A-13:** RFU of 5 variants and wild-type bacteria after 24 minute treatment

**Figure A-14:** Figure A-14: RFU of 5 variants and wild-type bacteria after 48 minute treatment

Based on the successful capturing protein expression results, we want to test the nanoparticle binding capability for future potential application. We incubated our construct variant bacteria, which are expressing NPs capturing protein, with nanoparticles and measured the fluorescence signal to quantify NPs (nanospheres with fluorescence) signals in the supernatant after centrifugation. The fluorescence delta change is determined by individually subtracting the absorbance readings of the negative control and construct variants, which both incubated with nanoparticle, to the positive control reading, which contains the same type and concentration of nanoparticle in phosphate buffer. The greater the delta change value is, the more efficient our capturing protein function is. In conclusion, OPHM and OPHT have consistently high Relative Fluorescence Unit (RFU) throughout 6, 12, 24 and 48 minutes incubation time from Figure A-11 to A-14. In terms of incubation time, 6 minutes is considered as the best incubation time length for bacteria to capture the most amount of nanoparticles from Figure A-11. The figures above show that compared to the wild-type bacteria, consistent in different time points, our bacteria with the surface nanoparticle capturing protein performs better.

![](https://static.igem.org/mediawiki/2019/8/85/T--UM_Macau--a15.png)

**Figure A-15:** Average standard curve for our yellow fluorescence nanoparticle

We made an average standard curve for the yellow fluorescence nanoparticle to convert Relative Fluorescence Unit (RFU) to concentration in parts per million unit (mg/L) for more straightforward data display. This was done to put the RFU into a more relatable for industrial application in the future.

![](https://static.igem.org/mediawiki/2019/8/8e/T--UM_Macau--a16_a17.jpeg)

![](https://static.igem.org/mediawiki/2019/0/06/T--UM_Macau--a18_a19.jpeg)

![](https://static.igem.org/mediawiki/2019/5/5a/T--UM_Macau--a20_a21.jpeg)

![](https://static.igem.org/mediawiki/2019/d/df/T--UM_Macau--a22_a23.jpeg)

![](https://static.igem.org/mediawiki/2019/b/b7/T--UM_Macau--a24_a25.jpeg)

**Figure A-16 to A-25:** Contrast of nanoparticle concentration (ppm) between in pellet and in supernatant collected from 5 construct variants with different incubation time length

By utilizing the same raw data of the capturing function, the relationship between the various incubation time points and capturing function were analyzed. After collecting the pellet of each bacteria culture, the trendlines show that all five bacteria construct included OPHT1, OPHT2, OPHT3, OPHM and OPHA have a significant increased capturing ability compared to the wild-type bacteria which may contain unspecific bindings. (Figure A-16, A-18, A-20, A-22 and A-24). The concentration of nanoparticle in each pellet of bacteria constructs has a significant increase which indicates that the capturing protein on the bacteria enhances its ability to capture more nanoparticles. The concentration of nanoparticle in the supernatant of all five bacteria constructs have more significant and drastic decrease compared to wild-type bacteria (Figure A-17, A-19, A-21, A-23, A-25). As the concentration of nanoparticles which are unbound to the capturing protein of the bacteria will remain in supernatant after centrifugation, thus the lesser nanoparticles left in the supernatant implies that there are more nanoparticles bound onto the capturing protein on the bacteria cell surface. These declining trendlines show that the observed increase in the concentration of nanoparticles in the pellet as shown in Figures A-16, A-18, A-20, A-22 and A-24 is reflected clearly as a decrease of the nanoparticle concentration in the supernatant as shown in Figures A-17, A-19, A-21, A-23, A-25. All the evidence mentioned above shows that all five capturing protein is successfully secreted and the capturing system is performing well. Thus, ouur bacteria SANCE is working well.

# Reference

1. Park, J. P., Choi, M.-J., Kim, S. H., Lee, S. H., & Lee, H. (2013). Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine Moiety. Applied and Environmental Microbiology, 80(1), 43–53. doi: 10.1128/aem.02223-13
2. Hong, H., Patel, D. R., Tamm, L. K., & Berg, B. V. D. (2006). The Outer Membrane Protein OmpW Forms an Eight-stranded β-Barrel with a Hydrophobic Channel. Journal of Biological Chemistry, 281(11), 7568–7577. doi: 10.1074/jbc.m512365200
3. Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. doi: 10.1016/j.addr.2012.09.039
4. Pei, J., Wen, S., Zhou, Y., Dong, Q., Liu, Z., Zhang, J., & Tian, W. (2011). A low band gap donor–acceptor copolymer containing fluorene and benzothiadiazole
5. Schwarz, D., Dötsch, V., & Bernhard, F. (2008). Production of membrane proteins using cell-free expression systems. Proteomics, 8(19), 3933–3946. doi: 10.1002/pmic.200800171

2nd Feature: Tyrosinase Secretion and Activation system

# I. Plasmid Construction

### PCR and Gel Running Results

![](https://static.igem.org/mediawiki/2019/f/ff/T--UM_Macau--b1.png)

**Figure B-1:** Size of Fragment NSP4-tyrosinase=1022bp

When have received our fragments from IDT. We digested the vector pBAD24 in order to directly ligate the received fragment. After gibson assembly, we have transformed the plasmid into the Stbl3 strain of bacteria and then picked 6 colonies and extracted the plasmid. After that, we amplified the NSP4-Tyrosinase DNA fragment of colonies 1, 2, 3, 4, 5 and 6 through PCR. From the gel run results above, we can see that all the bands’ size of NSP4-tyrosinase DNA fragments from all the colonies are at around 1000bp which is close to 1022bp which is what we expected. This result proves that we have successfully constructed pBAD24-NSP4.

### NSP4-tyrosinase plasmid’s sequencing result

![](https://static.igem.org/mediawiki/2019/e/e7/T--UM_Macau--b2.png)

**Figure B-2:** This is the aligned plasmid sequencing result. Two primers were used to check our gene of interest

Two flanking primers were used to overlap the gene of interest NSP4-Tyrosinase. From the plasmid sequencing results for NSP4-Tyrosinase colony 3 and 6, we can see that there are very few mismatched pairs in each forward and reverse primer pairs of each colony. But there are so many mismatch pairs in colony 5, therefore we didn’t use the plasmids from this colony to finish the further experiment. Colony 6 plasmid was chosen for the subsequent experiments.

# II. Protein Expression Test

### Western blotting results: NSP4-Tyrosinase induced and uninduced pellets

![](https://static.igem.org/mediawiki/2019/b/bc/T--UM_Macau--b3.png)

**Figure B-3:** This Western Blot result shows our pellet samples signal after lysis of the bacteria

We cultured NSP4-tyrosinase transformed bacteria, saved some volume of the culture as uninduced bacteria sample and induced the rest volume of bacteria culture with 0.2% arabinose for the next 6~8 hours. Next, we centrifuged both induced and uninduced samples, we saved both pellet and supernatant for target protein expression check using western blotting. We lysed the cells using a lysis buffer. However, in this first western blotting result as shown in Figure B-3, we were only able to clearly show that our target protein is expressed in the pellet sample. Tyrosinase which has the size of 34kDa has a strong detection signal compared to the uninduced samples. Although we got the right protein size, we want our protein to be secreted onto the supernatant. A possible explanation for this was that our protein concentration was not high enough in the supernatant sample thus we couldn’t see a signal for it.

### Western blotting results: Nickel pull-down of NSP4 supernatant

![](https://static.igem.org/mediawiki/2019/1/12/T--UM_Macau--b4.png)

**Figure B-4:** A. NSP4 arabinose induced (before pull-down)
B. NSP4 arabinose induced (after pull-down) C. bacteria transformed by vector without the insert(before pull-down). D. bacteria transformed by vector without the insert(after pull-down).

The results of Figure B-3 shows that there is not enough tyrosinase in the supernatant therefore we have performed Nickel pull-down. Figure B-4 above shows our results after pulling down the supernatant samples. It makes sense that our target protein is not detectable in either before (C sample) or after pull-down (D sample) in the supernatant from bacteria transformed with vector without tyrosinase fragment insert after centrifugation. By concentrating our protein of interest from the supernatant using pull-down assay (B sample), we were able to see, as shown in Figure B-4, the band of our target protein Tyrosinase, which is 34kDa in size. Indicating that it was pulled-down from the supernatant of induced samples while our target protein bands don’t show in the supernatant from bacteria transformed with tyrosinase gene without pull down. This result indicates that our protein is secreted in the extracellular space due to the NSP4-tyrosinase inserted in the pBAD24 vector backbone as it does not express in the bacteria sample which is only transformed with pBAD24 vector backbone without the insert.

# III. Functional Test

![](https://static.igem.org/mediawiki/2019/6/67/T--UM_Macau--b5.png)

**Figure B-5:** The bar chart above shows three information, arabinose concentration, Specific activity and time of incubation.

In order to investigate the best condition for activating the tyrosinase secretion system, we induced the E.coli with a series of concentrations of arabinose ranging from 0 to 133000μM as well as two different incubation times: 4 and 8 hours under each concentration. In Figure B-5, the tyrosinase activity increases while being induced by a higher concentration of arabinose, it reaches the highest tyrosinase activity when the concentration of arabinose added is the highest. However, longer incubation time would not contribute to a higher tyrosinase activity as the activity obviously does not increase much with 8 hour incubation even with very high arabinose concentration. Based on this functional test result, we can deem 133000μM arabinose induction with 4 hours incubation time with the induction chemical arabinose to be the best condition to collect the supernatant of bacteria culture and be used for further tests. With a higher concentration of arabinose induction, the expression of tyrosinase will increase in an appropriate incubation time length and would be able to show better enzyme function. The results that we have obtained shows that we are successful in secreting a functional tyrosinase enzyme out of our bacteria.

# Reference

1. Valipour, E., & Arikan, B. (2016). Increased production of tyrosinase from Bacillus megaterium strain M36 by the response surface method. Archives of Biological Sciences Arhiv Za Bioloske Nauke, 68(3), 659–668. doi: 10.2298/abs151002058v
2. Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli. AMB Express, 7(1). doi: 10.1186/s13568-017-0394-1
3. Guzman, L. M., Belin, D., Carson, M. J., & Beckwith, J. (1995). Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. Journal of Bacteriology, 177(14), 4121–4130. doi: 10.1128/jb.177.14.4121-4130.1995
4. Schierle, C. F., Berkmen, M., Huber, D., Kumamoto, C., Boyd, D., & Beckwith, J. (2003). The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the Escherichia coli Periplasm via the Signal Recognition Particle Pathway. Journal of Bacteriology, 185(19), 5706–5713. doi: 10.1128/jb.185.19.5706-5713.2003
5. Beckwith, J. (2013). The Sec-dependent pathway. Research in Microbiology, 164(6), 497–504. doi: 10.1016/j.resmic.2013.03.007
6. Agarwal, P., Singh, M., Singh, J., & Singh, R. (2019). Microbial Tyrosinases: A Novel Enzyme, Structural Features, and Applications. Applied Microbiology and Bioengineering, 3–19. doi: 10.1016/b978-0-12-815407-6.00001-0
7. Xu, D.-Y., Chen, J.-Y., & Yang, Z. (2012). Use of cross-linked tyrosinase aggregates as catalyst for synthesis of l-DOPA. Biochemical Engineering Journal, 63, 88–94. doi: 10.1016/j.bej.2011.11.009

3rd Feature: Magnetization and Control System

# I. Plasmid Construction

### PCR and Gel Running Results

![](https://static.igem.org/mediawiki/2019/e/e1/T--UM_Macau--c1.png)

**Figure C-1:** Gel running result for plasmid identity confirmation. Expected size of the FtnA fragment 574bp.

After performing gibson assembly of the FtnA fragment into the pBAD24 plasmid backbone we picked 3 colonies and then we extracted their plasmid and then we used PCR to amplify the FtnA DNA fragment. From the gel run results above, we can see that all the band size of FtnA DNA fragment from all the colonies are at around the expected size of 574 bp. This result shows that we have successfully constructed pBAD24-FtnA.

### FtnA plasmids sequencing result

![](https://static.igem.org/mediawiki/2019/4/44/T--UM_Macau--c2.png)

**Figure C-2:** Sequencing result for the pBAD24\_FtnA plasmid

After we have built the plasmid we sent them for sequencing. The sequencing results for FtnA colony 1, 2 and 3 are as shown in Figure C-2. We can see that there are very few mismatched pairs in the reverse primer of each colony. Therefore, all of them are usable. However, for our further experiments, we have used the plasmids from Colony #1 only.

# II. Protein Expression Test

### Western Blotting Results: Checking of FtnA Expression

![](https://static.igem.org/mediawiki/2019/a/a5/T--UM_Macau--c3.png)

**Figure C-3:** Western Blot result containing our protein bands pertaining to the FtnA size

Our FtnA gene was tagged with FLAG tag at the C-terminal. To confirm whether our FtnA protein is successfully expressed in our bacteria, we ran western blot with 7 protein samples which respectively extracted from 6 different bacteria colonies transformed with FtnA gene fragment inserted plasmid shown as above (colonies 1.1, 1.2, 1.3, 2.1, 2.2 and 2.3) and from bacteria without FtnA-plasmid transformation as our negative control in the western blot analysis (Figure C-3). We have used an anti-Flag primary antibody with HRP tag and used chemiluminescence to produce detectable signals. We can see that FtnA protein is successfully expressed in all 6 bacteria colonies compared to the negative control. Although there is variation in the amount of protein shown, this result tells us that after successful transformation we can be confident that the protein would be expressed regardless of which colony we choose.

### Western blotting results: FtnA expression level induced with different arabinose concentrations

![](https://static.igem.org/mediawiki/2019/7/71/T--UM_Macau--c4.png)

**Figure C-4:** Western Blot result containing our protein bands which we later analyzed

![](https://static.igem.org/mediawiki/2019/c/c9/T--UM_Macau--c5.png)

**Figure C-5:** Bar chart to represent the relative expression level and arabinose concentration

To investigate whether concentrations of arabinose will affect FtnA protein expression level, we ran western blot with bacteria lysis sample from BL-21 (DE3) bacteria incubated with 6 different concentrations of arabinose: 0-13300uM (Figure C-4). Band signal’s intensity from the membrane were analyzed using Image Studio. Normalized with internal control rpoB (which the protein size is 150kDa), we can see that FtnA protein expression level rises when the arabinose concentrations are increased (Figure C-5). Therefore, it indicates that the concentrations of arabinose can affect the FtnA protein expression level in the same bacteria strain. We have used both of these results for the characterization of the part number BBa\_K1679029 by OUC China 2015. This result would be a valuable characterization contribution to the said part for future teams who wishes to use the part and express the protein under the araBAD promoter.

### Western blotting results: FtnA expression level in different bacteria strains

![](https://static.igem.org/mediawiki/2019/f/f1/T--UM_Macau--c6.png)

**Figure C-6:** The figure shows the membrane containing our protein signal bands which we analyzed later

![](https://static.igem.org/mediawiki/2019/c/c6/T--UM_Macau--c7.png)

**Figure C-7:** The bar chart represents the different strains and their relative protein expression levels

Different E.coli strain types can influence FtnA protein expression levels. To investigate which E.coli strain is the best strain for Ftna protein expression, we ran western blot with the extracted protein samples from 6 different kinds of bacteria strains (Figure C-6). We used rpoB (DNA-directed RNA 9polymerase subunit beta) as an internal control for our western blot analysis. Band signal’s intensity from the membrane were analyzed using Image Studio. After being normalized against the internal control (rpoB), we can see that different protein expression levels could be observed in different bacteria strains and all BL-21 derivatives strains which includes Rosetta, Star, pLys and DE3 showed a much higher level of protein expression compared to wild-type BL-21 strain (WT) as well as Stbl3 and DH5α strains (Figure C-7). Therefore, we can conclude that BL-21 derivatives: Rosetta, Star, pLys and DE3 are suitable strains in expressing FtnA protein.

# III. Functional Test

![](https://static.igem.org/mediawiki/2019/4/42/T--UM_Macau--c8.png)

**Figure C-8:** This shows the magnetization test setup for checking the function of ftna protein.

After testing the protein expression level, we also want to know whether those expressed proteins are functional. A functional FtnA protein would increase the magnetization efficiency; we expect that after 1 hr, there would be significant migration of the bacteria toward the magnet. Therefore, we did a magnetization test: The pBAD24-FtnA construct was transformed respectively into the six different strains which are BL21 (Rosetta), BL-21 (Star), Stbl3, BL-21 (pLys), DH5 alpha and BL-21(DE3). Our negative control is the wild type BL-21 DE3 which is without the construct. Overnight cultures of 5 mL were made and after overnight culture they were all induced by 0.02% arabinose and cultured overnight. The next day, 1ml of 100mM ferric ammonium citrate for 24 hours was added. After incubation, we centrifuged the bacteria culture, resuspended it with autoclaved water and poured 2ml into the small dish. A round shape magnet was set under each small dish during the 1hour magnetization. In figure C-8, we can see that the strains, which includes BL-21 Rosetta, BL-21 Star, BL-21 pLys and BL-21 DE3 showed a clear bacteria migration towards the shape of the magnet after 1 hour. Meanwhile, no significant change can be observed in the wild type BL-21 strain (DE3) as well as Stbl3 and DH5α strains. This functional test results shows consistent results with our protein expression test result in Figure 4, and it also can demonstrate that our FtnA overexpression system can work in these strains that we would recommend using in the future.

# Reference

1. Lu, Y.-J., & Sun, M. (2014). A simple method for constructing magnetic Escherichia coli. doi: 10.1101/010249
2. Bauminger, E. R., & Nowik, I. (1989). Magnetism in plant and mammalian ferritin. Hyperfine Interactions, 50(1-4), 489–497. doi: 10.1007/bf02407681
3. Braun, V. (2003). Iron uptake by em Escherichia Coli em. Frontiers in Bioscience, 8(6). doi: 10.2741/1232
4. Bitoun, J. P., Wu, G., & Ding, H. (2008). Escherichia coli FtnA acts as an iron buffer for re-assembly of iron–sulfur clusters in response to hydrogen peroxide stress. BioMetals, 21(6), 693–703. doi: 10.1007/s10534-008-9154-7

# Discussion

Initially, we planned to combine all these three functional systems in our engineered E.coli. However, we were not able to achieve it due to insufficient time to perform the combination tests. We selected pET11a plasmid which has ribosome binding site (RBS) as vector backbone of nanoparticle capturing system, pBAD24 plasmid with NSP4 as vector backbone for tyrosinase secretion system and FtnA overexpression system respectively. pET11a and pBAD24 plasmid are both Ampicillin resistant as their origins of replication are compatible, we will not be able to select colonies which possess construction of all three features. Although we are able to do checking by using PCR technique whether the picked colony has three constructs, it will be very time consuming and laborious. Moreover, the incompatibility of plasmids is a statistical phenomenon, when one of the plasmids divides faster than others. They compete with each other for the replication machinery, thus we are not able to guarantee the ratio of our three final plasmid features as one might dominate over another. In addition, there will be only one plasmid would be passed down to the next generation after a couple of divisions, this can lead to heterogeneous population of bacteria in our culture. In order to maintain the homogeneous population during sub-culture, we must apply combined antibiotic for selection. Therefore, possible solutions are to reconstruct the system with compatible plasmids or to combine all three features into single plasmid by utilizing Golden Gate assembly. In this case, this plasmid may finally contains three different cloning sites and promoters for three features we suggested.

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