iGEM 2019 — Team VIT_Vellore

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# Team:VIT Vellore/Notebook

Team VIT Vellore/Description

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  [Description](https://2019.igem.org/Team%3AVIT_Vellore/Description)
  [Parts](https://2019.igem.org/Team%3AVIT_Vellore/Parts)
  [Design](https://2019.igem.org/Team%3AVIT_Vellore/Design)
  [Model](https://2019.igem.org/Team%3AVIT_Vellore/Model)
  [Experiments](https://2019.igem.org/Team%3AVIT_Vellore/Experiments)
  [Results](https://2019.igem.org/Team%3AVIT_Vellore/Results)
* [Safety](https://2019.igem.org/Team%3AVIT_Vellore/Safety)
* [Notebook](https://2019.igem.org/Team%3AVIT_Vellore/Notebook)
* [Characterization](https://2019.igem.org/Team%3AVIT_Vellore/Characterization)
* Human Practices

  [Overview](https://2019.igem.org/Team%3AVIT_Vellore/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AVIT_Vellore/Collaborations)
  [Integrated Human
  Practices](https://2019.igem.org/Team%3AVIT_Vellore/IHP)
  [Public Engagement
  and Outreach](https://2019.igem.org/Team%3AVIT_Vellore/Public_Engagement)
* Team

  [Members](https://2019.igem.org/Team%3AVIT_Vellore/Members)
  [Attributions](https://2019.igem.org/Team%3AVIT_Vellore/Attributions)
* [Awards](https://2019.igem.org/Team%3AVIT_Vellore/Awards)

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  [Experiments](https://2019.igem.org/Team%3AVIT_Vellore/Experiments)
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* [Safety](https://2019.igem.org/Team%3AVIT_Vellore/Safety)

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NOTEBOOK

## The experiments and processes involved

June

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| 3   Media Preperation  Media Preparation×  Prepared 100 mL LB broth in 2 flasks each and autoclaved it. | 4   Subculture  Subculture×  E. coli culture was obtained from general lab and thawed at room temperature.  100 μL of the culture was inoculated into the prepared LB broth.  Kept in the shaker incubator at 37°C for 24 hours. | 5   Streak  Streak×  2 LB plates were prepared and autoclaved.  The 24 hour culture was streaked on the LB plates. Control was kept blank. | 6   Contamination  Contamination×  Results  Contamination was observed in the streaked plates as well as the control plate.  The bacteria were overgrown.  Inferences    The bacteria reached the death phase in 24 hours incubation time. Therefore, incubation time needs to be reduced.  The culture obtained from general lab was not pure and hence, could not be used for further experiments. | 7   Re-culturing  Streak×  E. coli culture was obtained from research scholars in Dr. Gothandam’s lab.  T2 LB plates were prepared and autoclaved.  LB broth (100 mL) was prepared (2 flasks each) and autoclaved.  The E. coli culture was thawed at room temperature and 100 μL of it was inoculated in the LB broth. Control was kept blank.  The flasks were kept in shaker incubator at 37°C for 20 hours. | 8   Streak  Streak×  The 20 hour culture was streaked on the LB plates. Control was kept blank. | 9   Pure Culture  Streak×  Results  Not contamination observed in the streaked plate as well as the control.  The culture had grown adequately (no overgrowth).  Inferences    The bacteria were in the exponential phase of growth in 20 hours incubation time. Therefore, 20 hours incubation is sufficient to maintain viable cells for further experiments.  The culture obtained from Dr. Gothandam’s lab was pure and hence could be used for further experiments. |
| 10 | 11   Reagents Prepared | 12   Restriction Digestion  Restriction Digestion (works)×  Protocol: [Restriction Digestion](https://static.igem.org/mediawiki/2019/b/bd/T--VIT_Vellore--ProtoRestrictionDiges.pdf)  Results  Fragments of appropriate size were visualized on the gel.  Inferences  This indicates the restriction digestion protocol used works successfully.  The fragments of appropriate size obtained indicate that the restriction enzymes work successfully. | 13   Sub Culture | 14 | 15 | 16 |
| 17   Reagents Prepared  Reagents Prepared×  Reagents prepared for ligation.  Prepared buffers and autoclaved them and other consumables. | 18   Ligation  Ligation (fail)×  Results  The band observed in the gel was not of appropriate size.  Inferences  Ligation failed.  Reason (discovered later): Ligase Assay Buffer used for the experiment had been expired. | 19   Repeat Ligation  Repeat Ligation (works)×  Protocol: [Ligation](https://static.igem.org/mediawiki/2019/f/f6/T--VIT_Vellore--ProtoLigation.pdf)  Obtained Ligase Assay Buffer from Dr. Gothandam’s lab.  Results  The band visualized in the gel was of appropriate size.  Inferences  Ligation done successfully. | 20   Sub Culture | 21 | 22 | 23 |
| 24   Reagents Prepared  Reagents Prepared×  Reagents preparation for PCR.  Prepared PCR buffers and autoclaved them. | 25   PCR  PCR (fail)×  Results  Bands visualized were not of appropriate size.  The bands were not clear and were smeared.  The amplicons were not amplified enough and a very faint, thin band was observed.  Inferences  The dNTP volume added needs to be increased.  The agarose concentration in the gel needs to be increased to get a clear separation of the bands. | 26   Repeat PCR  Repeat PCR (works)×  Protocol: [PCR](https://static.igem.org/mediawiki/2019/2/2d/T--VIT_Vellore--ProtoPCR.pdf)  Results  Bands visualized were of appropriate size.  The bands were clear and not smeared.  The amplicons were amplified enough and a clear band was observed. | 27   Sub Culture | 28 | 29 | 30 |

July

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 2   Reagents Prepared  Reagents Prepared×  Reagents preparation for competent cell preparation.  Prepared reagents and buffers for competent cell preparation and autoclaved them and other consumables. | 3   Make a 24-hour culture. | 4   Competent Cell Preperation  Competent Cell Preperation×  Protocol: [Competent Cell Preperation](https://static.igem.org/mediawiki/2019/a/ab/T--VIT_Vellore--ProtoComCellPrep.pdf)  Performed competent cell preparation.  Results and Inferences  Competent cells were prepared successfully. | 5   Sub Culture | 6 | 7 |
| 8 | 9   Reagents Prepared  Reagents Prepared×  Reagents preparation for transformation.  Prepared media (LB and SOC) and autoclaved them along with consumables. | 10   Transformation  Transformation (fail)×  Results  No colonies growth was seen on the plate.  Inferences  Antibiotic concentration could be too high.  Transformation could be unsuccessful due to the plausible competent cells to be incompetent for successful transformation.  Another possibility could be that the protocol used was not appropriate for transformation. | 11   Repeat Transformation  Repeat Transformation (works)×  Protocol: [Transformation](https://static.igem.org/mediawiki/2019/4/40/T--VIT_Vellore--ProtoTransformation.pdf)  Transformation was repeated using a different protocol, with a lower antibiotic concentration.  Result  Isolated pure E. coli colonies were seen on the plates.  No contamination was observed.  Inferences  Antibiotic concentration used was appropriate.  The competent cells prepared in the previous experiment were competent enough for a successful transformation.  Protocol used was appropriate for a successful transformation. | 12   Sub Culture | 13 | 14 |
| 15   Reagents Prepared  Reagents Prepared×  Reagents preparation for plasmid DNA isolation.  Prepared the reagents and buffers for plasmid DNA isolation. | 16   Plasmid DNA Isolation  Plasmid DNA Isolation (fail)×  Results  The bands observed were not of appropriate size.  Inferences (probable reasons)  Concentration of reagents used in buffer preparations was not appropriate.  Ethidium bromide was probably not added.  Probable mistakes in buffer preparation. | 17   Repeat Plasmid DNA Isolation  Repeat Plasmid DNA Isolation (fail)×  Results  The bands observed were not of appropriate size.  RNA contamination was observed.  Inferences  Buffer pH was not appropriate since the buffers used were not freshly prepared.  RNAse A was not added. | 18   Fresh reagents made  Fresh reagents made×  Make overnight culture.  All reagents and buffers were re-prepared.  Culture was thawed at room temperature and inoculated into LB broth and incubated for 20 hours. | 19   Repeat Plasmid DNA Isolation  Repeat Plasmid DNA Isolation (works)×  Protocol: [Plasmid DNA Isolation](https://static.igem.org/mediawiki/2019/f/fd/T--VIT_Vellore--ProtoPlasDNA.pdf)  Transformation was repeated using a different protocol, with a lower antibiotic concentration.  Results  Bands observed were of appropriate size.  No RNA contamination was observed.  Inferences  The composition of the buffers and reagents was amended successfully.  The correct size of the bands indicate that the plasmid DNA has been successfully isolated.  Absence of contaminating bands indicate that the plasmid DNA obtained is pure and can be used for further characterization. | 20   Sub Culture | 21 |
| 22 | 23 | 24 | 25   Reagents Prepared  Reagents Prepared×  Reagents preparation for SDS PAGE.  Prepared all reagents and buffers for SDS PAGE. | 26   SDS Page  SDS Page (fail)×  Results  Experiment failed. Bands were not separated successfully.  Inferences (probable reasons)  Acrylamide: bis-acrylamide ratio used was inappropriate.  Mistakes in reagent and/or buffer preparation. | 27   SDS Page  Repeat SDS Page (fail)×  Results  The gel was not destained properly.  Inferences  Staining and/or destaining time was not correct. (Destaining time was less). | 28   Make fresh reagents. |
| 29   SDS Page  SDS Page (works)×  Protocol: [SDS Page](https://static.igem.org/mediawiki/2019/6/67/T--VIT_Vellore--ProtoSDSPage.pdf)  Results  Bands were separated successfully.  Gel was destained successfully.  Inferences  The protocol used was appropriate.  Staining and destaining time was correct.  Reagent and buffer preparation was correct. | 30   Sub Culture | 31   SDS Page (works) |  |  |  |  |

August

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | 1 | 2 | 3 | 4 |
| 5   Reagents assembling and autoclaving for alpha amylase experiment. | 6   Alpha Amylase  Alpha Amylase Experiment×  C-source, Organic and inorganic nitrogen.  Protocols: [Alpha Amylase Characterization](https://static.igem.org/mediawiki/2019/a/aa/T--VIT_Vellore--ProtoAlphaAmylase.pdf)  Results  Relative expression levels of alpha-amylase are compared under different physicochemical conditions. | 7   Sub Culture | 8 | 9 | 10 | 11 |
| 12 | 13 | 14 | 15 | 16   Replication of restriction digestion (works) | 17   Replicate of ligation (works) | 18   Replicate of PCR (works) |
| 19   Replicate of competent cell preparation (works) | 20   Replicate of electroporation (works) | 21   Replicate of PCR (works) | 22   Replicate of plasmid DNA isolation (works) | 23   Sub Culture | 24 | 25 |
| 26   Get ordered construct | 27 | 28 | 29 | 30   Sub Culture | 31 |  |

September

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  |  | 1 |
| 2   PCR  PCR×  Amplification of both the fragments of the construct separately.  Results  PCR worked successfully. The bands visualized in the gel were of appropriate size.  The bands were quite thick, indicating amplification. | 3   PCR Clean-up  PCR Clean-up×  Used commercially available PCR clean-up kit for PCR clean-up.  Results  Only the amplified DNA is left in the clean-up while the unamplified DNA, leftover dNTPs, MgCl2, etc. is washed up. | 4   Nanodrop  Nanodrop×  Performed to obtain the concentration of the amplified PCR product after PCR clean-up.  PCR clean-up reagent was used as the blank.  Results  The concentration of the PCR amplicon obtained was ~200 μg.  Inference  PCR amplification is successfully since the concentration of the original DNA template used for PCR was ~2 μg. | 5   Sub Culture | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14   Sub Culture | 15   Restriction Digestion  Restriction Digestion×  Restriction digestion was performed of the plasmid backbone and the antisense construct.  The Xba I and Spe I sites on both fragments would have sticky ends.  Results  The fragments visualized on the gel were of appropriate sizes (antisense and backbone).  Inference  The restriction enzymes worked successfully.  The fragments have sticky ends generated for ligation. |
| 16   Ligation  Ligation×  Ligation performed of the antisense construct with the plasmid backbone.  Results  The band visualized on the gel was of appropriate size (size of antisense + backbone together).  Inferences  The fragments were ligated successfully to give a single sequence.  This sequence can be transformed further to competent E. coli cells to check for bacterial growth in presence of combination of different antibiotic resistances. | 17 | 18 | 19 | 20   Sub Culture | 21   Competent Cell Preperation  Competent Cell Preperation×  Competent cells were prepared and stored at -80°C.  Result and Inference  The competent cells are capable of transformation. | 22   Transformation - Part I  Transformation - Part I×  Transformation of ampicillin resistant pBR322 in E coli cells.  The E. coli competent cells were transformed with ampicillin resistant pBR322 plasmid by heat shock method.  The control had non-transformed E. coli competent cells on ampicillin plates.  Results  The test plate had bacterial colonies growing.  No contamination was observed.  The control plates had no colonies growing.  Inferences  Transformation is successful since the test plate shows growth.  The plasmid containing ampicillin resistance confers selective pressure to the bacteria to retain the plasmid in ampicillin resistant plates.  Non-transformed E. coli cells do not have ampicillin resistance, therefore, there in cell death in control. |
| 23 | 24 | 25 | 26   Transformation - Part II  Transformation - Part II×  Transformation of our ligated construct into E. coli cells.  E. coli competent cells transformed with ligated plasmid construct containing Chloramphenicol resistance by heat shock method.  Control had non-transformed E. coli competent cells on chloramphenicol plates.  Results  The test plate had bacterial colonies growing.  No contamination was observed.  The control plates had no colonies growing.  Inferences  Transformation is successful since the test plate shows growth.  The plasmid containing chloramphenicol resistance confers selective pressure to the bacteria to retain the plasmid in chloramphenicol resistant plates.  Non-transformed E. coli cells do not have chloramphenicol resistance, therefore, there in cell death in control. | 27   Sub Culture | 28 | 29   Transformation - Part III  Transformation - Part III×  Transformation of our construct plasmid (containing chloramphenicol resistance) and pBR322 into E. coli competent cells.  Plates contain chloramphenicol and tetracycline.  Control had non-transformed E. coli competent cells on Chloramphenicol + Tetracycline plates.  Results  The test plate had bacterial colonies growing.  No contamination was observed.  The control plates had no colonies growing.  Inferences  Transformation is successful since the test plate shows growth.  Chloramphenicol + Tetracycline resistance confers selective pressure to the bacteria to retain the plasmid in ampicillin resistant plates.  Non-transformed E. coli cells do not have chloramphenicol resistance, therefore, there in cell death in control.  The transformed cells from this experiment as referred to as ‘double transformants’. |
| 30 |  |  |  |  |  |  |

October

| Monday | Tuesday | Wednesday | Thursday | Friday | Saturday | Sunday |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3   Sub Culture | 4   Validation  Repeat Validation×  Plating double transformants containing Chloramphenicol and Tetracycline resistances into Ampicillin plates.  Control had non-transformed E. coli competent cells on (Chloramphenicol + Tetracycline + Ampicillin) plates.  Results  No growth on test plate. No contamination was observed. The control plates had no colonies growing.  Inference  E. coli cells do not have Chloramphenicol, Tetracycline and Ampicillin resistance natively, therefore control shows no growth. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance and incorporation of pBR322 into the E. coli confers them with Tetracycline and Ampicillin resistance. Therefore, cells transformed with our construct along with pBR322 should survive Chloramphenicol as well as Tetracycline. But, since antisense is also incorporated as a part of our construct which inhibits the Ampicillin resistance gene present in pBR322, thereby resensitizing to the bacteria to Ampicillin. Therefore, the cells do not survive due to Ampicillin present in the plates and the inhibited Ampicillin resistance gene. | 5 | 6 || 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  | 28 |  |

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# Team:VIT Vellore/Experiments

Team VIT Vellore/Description

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* Project

  [Description](https://2019.igem.org/Team%3AVIT_Vellore/Description)
  [Parts](https://2019.igem.org/Team%3AVIT_Vellore/Parts)
  [Design](https://2019.igem.org/Team%3AVIT_Vellore/Design)
  [Model](https://2019.igem.org/Team%3AVIT_Vellore/Model)
  [Experiments](https://2019.igem.org/Team%3AVIT_Vellore/Experiments)
  [Results](https://2019.igem.org/Team%3AVIT_Vellore/Results)
* [Safety](https://2019.igem.org/Team%3AVIT_Vellore/Safety)
* [Notebook](https://2019.igem.org/Team%3AVIT_Vellore/Notebook)
* [Characterization](https://2019.igem.org/Team%3AVIT_Vellore/Characterization)
* Human Practices

  [Overview](https://2019.igem.org/Team%3AVIT_Vellore/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AVIT_Vellore/Collaborations)
  [Integrated Human
  Practices](https://2019.igem.org/Team%3AVIT_Vellore/IHP)
  [Public Engagement
  and Outreach](https://2019.igem.org/Team%3AVIT_Vellore/Public_Engagement)
* Team

  [Members](https://2019.igem.org/Team%3AVIT_Vellore/Members)
  [Attributions](https://2019.igem.org/Team%3AVIT_Vellore/Attributions)
* [Awards](https://2019.igem.org/Team%3AVIT_Vellore/Awards)

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  [Description](https://2019.igem.org/Team%3AVIT_Vellore/Description)
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  [Design](https://2019.igem.org/Team%3AVIT_Vellore/Design)
  [Model](https://2019.igem.org/Team%3AVIT_Vellore/Model)
  [Experiments](https://2019.igem.org/Team%3AVIT_Vellore/Experiments)
  [Results](https://2019.igem.org/Team%3AVIT_Vellore/Results)

* [Safety](https://2019.igem.org/Team%3AVIT_Vellore/Safety)

* [Notebook](https://2019.igem.org/Team%3AVIT_Vellore/Notebook)

* [Characterization](https://2019.igem.org/Team%3AVIT_Vellore/Characterization)

* Human Practices ▼

  [Overview](https://2019.igem.org/Team%3AVIT_Vellore/Human_Practices)
  [Collaborations](https://2019.igem.org/Team%3AVIT_Vellore/Collaborations)
  [IHP](https://2019.igem.org/Team%3AVIT_Vellore/IHP)
  [Public Engagement](https://2019.igem.org/Team%3AVIT_Vellore/Public_Engagement)

* Team ▼

  [Members](https://2019.igem.org/Team%3AVIT_Vellore/Members)
  [Attributions](https://2019.igem.org/Team%3AVIT_Vellore/Attributions)

* [Awards](https://2019.igem.org/Team%3AVIT_Vellore/Awards)

EXPERIMENTS

## The experiments conducted

## Protocols

---

[Restriction Digestion](https://static.igem.org/mediawiki/2019/b/bd/T--VIT_Vellore--ProtoRestrictionDiges.pdf)

[Ligation](https://static.igem.org/mediawiki/2019/f/f6/T--VIT_Vellore--ProtoLigation.pdf)

[PCR](https://static.igem.org/mediawiki/2019/2/2d/T--VIT_Vellore--ProtoPCR.pdf)

[Competent Cell Preperation](https://static.igem.org/mediawiki/2019/a/ab/T--VIT_Vellore--ProtoComCellPrep.pdf)

[Transformation](https://static.igem.org/mediawiki/2019/4/40/T--VIT_Vellore--ProtoTransformation.pdf)

[Plasmid DNA Isolation](https://static.igem.org/mediawiki/2019/f/fd/T--VIT_Vellore--ProtoPlasDNA.pdf)

[SDS PAGE](https://static.igem.org/mediawiki/2019/6/67/T--VIT_Vellore--ProtoSDSPage.pdf)

[DNS Assay (Characterization)](https://static.igem.org/mediawiki/2019/a/aa/T--VIT_Vellore--ProtoAlphaAmylase.pdf)

## Experiments

---

#### Overview

The following experiments had been performed with the view of validating our designed construct. The plasmid backbone that we used had chloramphenicol resistance. Our designed construct and plasmid backbone were digested with specific restriction enzymes to obtain sticky ends, and were further ligated using T4 DNA ligase. Double transformation of competent E.Coli cells with pBR322 plasmid (imparting tetracycline and ampicillin resistance) and assembled plasmid construct (imparting chlorampheniciol resistance) was done by heat shock method. The successful double transformation was verified by plating the competent cells on tetracycline + chloramphenicol LB agar and observing E.Coli growth after incubation.

The validation was done by:

* Plating the double transformed cells on ampicillin + tetracycline + chloramphenicol LB agar – Low growth was observed on this plate.

* Sub-culturing the double transformed cells from tetracycline + chloramphenicol LB agar and plating the cells on tetracycline + chloramphenicol as well as ampicillin + tetracycline +chloramphenicol LB agar plates – Growth was observed on the tetracycline + chloramphenicol plate while comparatively low growth was observed on the ampicillin + tetracycline + chloramphenicol plate.

Both the validations indicated that the ampicillin resistance which was incorporated had been destroyed, thereby causing cell death due to ampicillin. Thus, proving the successful function of our designed construct.

#### Experiments

Amplification of the constructs

* We performed PCR to amplify the constructs.

* The presence of the amplified DNA was confirmed by agarose gel electrophoresis.

* After PCR, the amplified DNA was purified and the impurities were separated by PCR clean-up.

Restriction digestion of plasmid backbone and antisense construct

* The plasmid backbone and the antisense construct was subjected to double restriction digestion by XbaI and SpeI for 1 hour at 37oC.

* This would result in the formation of sticky ends at XbaI site and SpeI site on both the DNA fragments.

Ligation of antisense construct and plasmid backbone

* The digested antisense construct and plasmid backbone were ligated at 16 oC for 2 hours, using T4 DNA ligase.

* After ligation, the ligated construct was verified by agarose gel electrophoresis.

Preparation of competent host cells

* Overnight grown E.Coli DH5α culture was pelleted and re-suspended in 100 mM MgCl2.

* The suspension was re-pelleted and suspended in 100 mM CaCl2.

* The suspension was again pelleted and suspended in 85mM CaCl2 + 15% glycerol.

* The final suspension was snap-freezed in liquid nitrogen and stored at -80oC.

Transformation of ampicillin + tetracycline resistant pBR322 into E.Coli cells

* E.Coli DH5α competent cells were thawed.

* The E.Coli competent cells were transformed with ampicillin + tetracycline resistant pBR322 plasmid by heat shock method.

* The transformed cells were plated on LB agar plates containing ampicillin and tetracycline, followed by incubation.

* A control setup was prepared by plating non-transformed E.Coli competent cells on ampicillin-containing agar plates.

* Growth of E.Coli cells on the test plate was observed, while the control setup did not show any E.Coli growth.

Purpose

* In native state, E.Coli DH5α cells do not have resistance to ampicillin and tetracycline, thereby the cells in control setup would undergo cell death.

* However, growth of E.Coli cells in the test setup was the result of development of ampicillin and tetracycline resistance in the cells, thereby indicating successful transformation of the pBR322 plasmid into the E.Coli cells.

Transformation of our ligated construct into E.Coli cells

* E.Coli DH5α competent cells were thawed.

* The E.Coli competent cells were transformed with ligated plasmid construct (plasmid backbone contains chloramphenicol resistance gene) by heat shock method.

* The transformed cells were plated on LB agar plates containing chloramphenicol, followed by incubation.

* A control setup was prepared by plating non-transformed E.Coli competent cells on chloramphenicol-containing agar plates.

* Growth of E.Coli cells on the test plate was observed, while the control setup did not show any E.Coli growth.

Purpose

* In native state, E.Coli cells do not have resistance to chloramphenicol, thereby the cells in control setup would undergo cell death.

* However, growth of E.Coli cells in the test setup was the result of development of chloramphenicol resistance in the cells, thereby indicating successful transformation of the construct plasmid into the E.Coli cells.

Transformation of construct plasmid and pBR322 into E.Coli cells

* E.Coli DH5α competent cells were thawed.

* The E.Coli competent cells were transformed with our construct (chloramphenicol resistant) plasmid and pBR322 plasmid by heat shock method.

* The transformed cells were plated on LB agar plates containing chloramphenicol and tetracycline, followed by incubation.

* A control setup was prepared by plating non-transformed E.Coli competent cells on agar plates containing both tetracycline and chloramphenicol.

* Growth of E.Coli cells on the test plate was observed, while the control setup did not show any E.Coli growth.

Purpose

* n native state, E.Coli cells do not have resistance to chloramphenicol or tetracycline, thereby the cells in control setup would undergo cell death.

* However, growth of E.Coli cells in the test setup was the result of development of resistance to both tetracycline and chloramphenicol in the cells, thereby indicating successful transformation of both, the pBR322 plasmid and the construct plasmid into the E.Coli cells.

* For simplification, the transformed cells obtained here would be referred to as double transformed cells.

#### Construct Verification

Plating double transformed cells on triple antibiotic plate

* LB agar plates containing ampicillin, tetracycline and chloramphenicol was prepared.

* The LB agar plate was plated with the double transformed cells, and the agar plate was incubated.

* Low growth of E.Coli cells was observed on the agar plate.

Purpose

* The low growth on the agar plate indicates cell death due to inactivation of the ampicillin resistance gene by the antisense part of our construct.

Sub-culturing of double transformed cells from chloramphenicol + tetracycline plates

* Two LB agar plates, one containing Chloramphenicol and Tetracycline (Plate A) and the other containing Chloramphenicol, Ampicillin, and Tetracycline (Plate B) were prepared.

* A single colony was picked from the overnight grown chloramphenicol + tetracycline plates and grown in LB Broth containing Chloramphenicol and Tetracycline, overnight.

* The next day, the overnight broth culture was plated on Plate A and Plate B, and both the plates were incubated.

* Growth was observed on plate A and comparatively lesser growth was observed on plate B.

Purpose

* The low growth on the agar plate B indicates cell death due to presence of ampicillin, as the cells grew on the plate A, which contained the other antibiotics, except ampicillin.

* This indicates cell death due to inactivation of the ampicillin resistance gene by the antisense construct.

#### Conclusion

* Transformation of both the plasmids (pBR322 and our construct), would impart resistance to the bacteria against ampicillin, tetracycline as well as chloramphenicol.

* The double transformed cells were able to grow on the LB agar plates containing tetracycline and chloramphenicol, due to presence of tetracycline and chloramphenicol resistance..

* However, since our genetic construct was designed to inactivate the ampicillin resistance gene, when the double transformed cells was plated on the LB agar plate, due to inactivation of the ampicillin resistance gene, the cells were killed due to the presence of ampicillin in the LB agar plate.

* Thus, the above observation would indicate that our designed construct has successfully counteracted the antibiotic resistance towards ampicillin, thereby validating the purpose of our construct.

## Development of the Experiments

---

Optimization

We started off our experimentation process with optimizing protocols for our experimentation. We started off by implementing small changes to the protocols for competent cell preparation, transformation, Agarose gel electrophoresis, Bacterial lysis methods and RNA isolation methodologies to ensure that they work optimally in the high heat and humidity conditions of the southern subcontinent.

SELEX To Antisense

SELEX was one of the first methods we considered to combat antibiotic resistance. This is a very new method designed to emulate RNA silencing. The reason we decided against this method further in our project is because, this method is very non-specific. This short sequence of amino acids will bind to any RNA sequence comparable in binding energy to the sequence it was designed for. Thus, while looking for alternatives, we decided to do with RNA silencing using a viral gene insert. This is specific to the sequence it is designed for and can be muted when necessary.

Phage to Plasmid

Working with bacteriophages requires access to a lab with the safety clearances from concerned authorities. While we had originally planned to conduct experiments using bacteriophages directly, we could not obtain access to such a lab.

So, we proceeded with a change in our plans, and decided to work with plasmids, which we use to introduce our construct into a bacteria, inside which we are able to control the selective expression of J proteins, and have control over activation of the lytic and lysogenic cycle as per need.

Plasmid Change

We started off with the vision of incorporating all our construct into one plasmid. The construct was too large to be accommodated in the plasmid with selectable markers (here pBR322). So we decided on a 2 plasmid system to efficiently transfer our DNA construct to a bacterium. This enabled us to transfer a larger volume of DNA while preserving the selectable markers.

Multiple Step Transformation To 1 Step Double Transformation

In our experimental design, we had earlier planned to do a step by step double transformation where we first transform the competent cells with plasmid 1 and then add the marker antibiotic to make sure selective pressure is maintained. After this, we would again make the cells competent for transformation and transform the cells with plasmid 2. This step by step transformation would have taken a lot more time and moreover composition of antibiotics in the petri-plates we made was such that we could directly do a double transformation and by platting it, we would get to know the results. By making this change, we were also able to save a lot of time as step by step transformation would mean performing competent cell preparation twice.

Changes in method for transferring colonies from plate 4 to plate 5 in the final experiment design

In our final experiment design, plate 4 contains two antibiotics, Chloramphenicol as well as Tetracycline. This means that only double transformants can survive on this plate and all colonies will contain both plasmid.

For testing our construct, we needed to plate colonies from plate 4 onto plate 5 which contains ampicillin in addition to the other two antibiotics and lower growth on plate 5 would confirm the working of our construct. We made two iterations to the experiment design in this step:

1. Initially we planned to pick up a colony from plate 4 using inoculum loop and directly place it in plate 5 without sub culturing. However, if we did this, comparing the growth patterns in both the plates would become a problem due to culture on plate 4 being non-homogeneous in nature.
2. Earlier we planned that plate 5 would only contain Ampicillin and not the other two antibiotics however in such a scenario, there is always a chance of plasmid rejection due to no use of the plasmid hence for maintaining selective pressure, we went ahead with plate 5 containing all the three antibiotics.

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# Team:VIT Vellore/Results

Team VIT Vellore/Description

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  [Description](https://2019.igem.org/Team%3AVIT_Vellore/Description)
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  [Design](https://2019.igem.org/Team%3AVIT_Vellore/Design)
  [Model](https://2019.igem.org/Team%3AVIT_Vellore/Model)
  [Experiments](https://2019.igem.org/Team%3AVIT_Vellore/Experiments)
  [Results](https://2019.igem.org/Team%3AVIT_Vellore/Results)

* [Safety](https://2019.igem.org/Team%3AVIT_Vellore/Safety)

* [Notebook](https://2019.igem.org/Team%3AVIT_Vellore/Notebook)

* [Characterization](https://2019.igem.org/Team%3AVIT_Vellore/Characterization)

* Human Practices ▼

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  [IHP](https://2019.igem.org/Team%3AVIT_Vellore/IHP)
  [Public Engagement](https://2019.igem.org/Team%3AVIT_Vellore/Public_Engagement)

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  [Attributions](https://2019.igem.org/Team%3AVIT_Vellore/Attributions)
* [Awards](https://2019.igem.org/Team%3AVIT_Vellore/Awards)

RESULTS

## The results of the experiments performed

## Introduction

---

Our final assembled construct contains resistance against Chloramphenicol (pSB1C3, backbone). The inserted fragment contains antisense sequence against Ampicillin resistance gene present in pBR322. pBR322 contains resistance against Ampicillin and Tetracycline.

Bacteria are transformed with:

1. pBR322, conferring resistance to Ampicillin
2. Assembled construct conferring resistance to Chloramphenicol
3. Double transformants containing both pBR322 and our assembled construct. The bacterial colonies can survive Tetracycline and Chloramphenicol antibiotics but are sensitive to Ampicillin (due to the antisense sequence against Ampicillin resistance present in our construct).

## Assembly of the required construct

---

The linearized backbone, pSB1C3 2070 bp, is ligated to our insert (designed composite fragment) (483 bp) following Restriction Digestion (using XbaI and SpeI). The insert was initially amplified using PCR. Agarose Gel Electrophoresis was performed to confirm the assembly of the desired plasmid.

![](https://static.igem.org/mediawiki/2019/7/7e/T--VIT_Vellore--ResultsLeftGel.jpg)

PCR Amplification of part

![](https://static.igem.org/mediawiki/2019/6/6d/T--VIT_Vellore--ResultsRightGel.jpg)

Confirmation of successful cloning

## Transformation Results

---

#### Transformation of Ampicillin resistant pBR322 into E. coli cells

E. coli cells were transformed with pBR322 plasmid containing Ampicillin and Tetracycline resistance and plated on LB plates containing Ampicillin. Control plate had non-transformed E. coli competent cells on Ampicillin-containing plates.

![](https://static.igem.org/mediawiki/2019/0/00/T--VIT_Vellore--ResultsA.jpg)

Results:

1. Test plates showed bacterial growth.
2. No growth observed on control plate.

Inference:

1. E. coli cells do not have Ampicillin resistance natively, therefore control shows no growth.
2. Incorporation of pBR322 plasmid into E. coli cells confers them with Ampicillin resistance. Therefore, cells transformed with pBR322 survive Ampicillin.
3. Conferring Ampicillin resistance to E. coli enables us to further validate our construct having antisense against the Ampicillin resistance gene.

#### Transformation of our construct (containing Chloramphenicol resistance and antisense against Ampicillin) into E. coli cells

E. coli cells were transformed with our construct containing Chloramphenicol resistance and plated on LB plates containing Chloramphenicol. Control plate had non-transformed E. coli competent cells on Chloramphenicol-containing plates.

![](https://static.igem.org/mediawiki/2019/9/90/T--VIT_Vellore--ResultsC.jpg)

Results:

1. Test plates showed bacterial growth.
2. No growth observed on control plate.

Inference:

1. E. coli cells do not have Chloramphenicol resistance natively, therefore control shows no growth.
2. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance. Therefore, cells transformed with our construct survive Chloramphenicol.
3. Conferring Chloramphenicol resistance to E. coli enables us to confirm that our construct (containing antisense to Ampicillin resistance gene) has been successfully incorporated into the E. coli cells.

#### Transformation of E. coli cells with our construct (containing Chloramphenicol resistance and antisense against Ampicillin) + pBR322 (containing Ampicillin and Tetracycline resistance)

E. coli cells were transformed with our construct (containing Chloramphenicol resistance and antisense against Ampicillin resistance gene) along with pBR322 (containing Ampicillin and Tetracycline resistance) and plates on LB plates containing Chloramphenicol and Tetracycline. Control plate had non-transformed E. coli competent cells on Chloramphenicol and Tetracycline containing plates.

We refer these cells as ‘double transformants’ in further experiments.

![](https://static.igem.org/mediawiki/2019/d/d6/T--VIT_Vellore--ResultsC%2BT2.jpg)

Results:

1. Test plates showed bacterial growth.
2. No growth observed on control plate.

Inference:

1. E. coli cells do not have Chloramphenicol and Tetracycline resistance natively, therefore control shows no growth.
2. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance and incorporation of pBR322 into the E. coli confers them with Tetracycline and Ampicillin resistance. Therefore, cells transformed with our construct along with pBR322 survive Chloramphenicol as well as Tetracycline.
3. Cells surviving on these plates have our construct having the antisense to the beta-lactamase (Ampicillin resistance) gene present in pBR322.

## Validation of our Construct

---

#### Plating double transformants on (Amp + Tet + Chl) plates

Plating double transformants on (Amp + Tet + Chl) plates:

The double transformants containing pBR322 (containing Ampicillin and Tetracycline resistance) along with our construct (containing Chloramphenicol resistance and antisense against Ampicillin resistance gene) were picked up from the previous Chl+Tet plate and were plated on plates containing Ampicillin, Tetracycline and Chloramphenicol.

Control plate had non-transformed E. coli competent cells on Ampicillin, Tetracycline and Chloramphenicol containing plates.

![](https://static.igem.org/mediawiki/2019/f/fb/T--VIT_Vellore--ResultsA%2BC%2BT2.jpg)

Results:

1. Low growth observed on test plate.
2. No growth observed on control plate.

Inference:

1. E. coli cells do not have Chloramphenicol, Tetracycline and Ampicillin resistance natively, therefore control shows no growth.
2. Incorporation of our construct into E. coli cells confers them with Chloramphenicol resistance and incorporation of pBR322 into the E. coli confers them with Tetracycline and Ampicillin resistance. Therefore, cells transformed with our construct along with pBR322 should survive Chloramphenicol as well as Tetracycline. But, since antisense is also incorporated as a part of our construct which inhibits the Ampicillin resistance gene present in pBR322, thereby resensitizing to the bacteria to amplicillin. Therefore, there is very low growth due to Ampicillin present in the plates and the inhibited Ampicillin resistance gene.

#### Growing double transformants in LB broth under selective pressure and plating them on (Chl + Tet) and (Amp + Chl + Tet) plates

The double transformants containing pBR322 (containing Ampicillin and Tetracycline resistance) along with our construct (containing Chloramphenicol resistance and antisense against Ampicillin resistance gene) were inoculated in LB broth containing Chloramphenicol and Tetracycline (selective pressure).

The cells from the broth were then plated on:

1. Chloramphenicol + Tetracycline (Chl + Tet) containing plates
2. Chloramphenicol + Tetracycline + Ampicillin (Chl + Tet + Amp) containing plates

![](https://static.igem.org/mediawiki/2019/a/a7/T--VIT_Vellore--ResultsC%2BT.jpg)

![](https://static.igem.org/mediawiki/2019/e/e9/T--VIT_Vellore--ResultsA%2BC%2BT.jpg)

Results:

1. Growth observed on (Chl + Tet) plate was higher than the initial growth observed after transformation on (Chl + Tet) plates.
2. Low growth was observed on the (Chl + Tet + Amp) plate.

Inference:

1. LB broth gives selective pressure for the double transformants (containing our construct + pBR322) to retain both the plasmids.
2. Sub-cultured bacteria containing our construct along with pBR322 are more pure when replated on (Chl + Tet) plates. Therefore, the growth observed is higher on cells plated after sub-culturing in the (Chl + Tet) plate when compared to the growth observed on the initial (Chl + Tet) plate.
3. Low growth is observed on (Chl + Tet + Amp) plate indicates successful integration of our construct containing antisense to Ampicillin resistance which inhibits the Ampicillin resistance gene, thereby, preventing growth in presence of Ampicillin.

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