iGEM 2016 — Team IIT_Kharagpur

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# Team:IIT Kharagpur/Notebook

IGEM-IIT Kharagpur- Notebook

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# NOTEBOOK

![](https://static.igem.org/mediawiki/2016/c/cd/T--IIT_Kharagpur--notebook.png)

July*2016*

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* [20](#20july)
* [21](#21july)
* [22](#22july)
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* [25](#july25)
* [26](#july26)
* [27](#july27)
* [28](#july28)
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August*2016*

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* [1](#august1)
* [2](#august2)
* [3](#august3)
* [4](#august4)
* 5
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* [7](#august7)

* [8](#august8)
* [9](#august9)
* [10](#august10)
* [11](#august11)
* [12](#august12)
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* [16](#august16)

- [17](#august17)
- [18](#august18)
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* [22](#august22)
* [23](#august23)
* [24](#august24)
* [25](#august25)
* [26](#august26)
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* [29](#august29)
* 30
* [31](#august31)
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September*2016*

* Mon
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* Wed
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* 29
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* [1](#september1)
* [2](#september2)
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* 5
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* [8](#september8)
* [9](#september9)
* [10](#september10)
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* [12](#september12)
* [13](#september13)
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* [19](#september19)
* [20](#september20)
* [21](#september21)
* [22](#september22)
* [23](#september23)
* [24](#september24)
* [25](#september25)

* [26](#september26)
* [27](#september27)
* [28](#september28)
* [29](#september29)
* [30](#september30)
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* 3
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October*2016*

* Mon
* Tue
* Wed
* Thu
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* Sat
* Sun

* 26
* 27
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* 29
* 30
* [1](#october1)
* [2](#october2)

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## WEEK ONE - ( 20th July - 22th July ) 20th july, Wednesday DH5 alpha competent cells were prepared for transformation experiments. 21st July, Thursday Transformation of the prepared competent cells was carried out. The plasmids used for transformation were YFP (BBa\_K592101), HIV cleavage site (BBa\_I712015), and the protease construct (BBa\_K743013) 22nd July, Friday Replication plating for above transformed parts was done. This including the three sets of transformed cells containing the plasmids YFP (BBa\_K592101), the HIV cleavage site (BBa\_I712015), and the protease construct (BBa\_K743013).

## WEEK TWO - ( 25th July - 30th July ) 25th july, Monday 1 litre of LB media was prepared and duly autoclaved. 5 plates each of the antibiotics chloramphenicol, ampicillin and ampicillin+Kanamycin were prepared. 26th July, Tuesday 20ml Cultures were prepared for the plasmid extraction of each of YFP (BBa\_K592101), the HIV cleavage site (BBa\_I712015) and the protease construct (BBa\_K743013). 27th July, Wednesday Plasmid extraction was done for YFP (BBa\_K592101), the HIV cleavage site (BBa\_I712015), and the protease construct (BBa\_K743013) using mini-prep plasmid DNA extraction kit. 28th July, Thursday DNA quantification was done using Nano Drop. YFP: 250 ng/ul Protease: 170 ng/ul Cleavage Site: 120 ng/ul 1% agarose gel was prepared for screening of the plasmids extracted by the mini prep kit. The FRET construct was kept for overnight digestion at the restriction sites E and P. Digestions with E and P for YFP, Protease and Cleavage Site | | YFP | Protease | Cleavage Site | | --- | --- | --- | --- | | Component | Volume(ul) | Volume(ul) | Volume(ul) | | DNA | 2 | 3 | 3.5 | | 2.1 Buffer | 2 | 2 | 2 | | ECoRI | 0.5 | 0.5 | 0.5 | | Psti | 0.5 | 0.5 | - | | Nf H2O | 15 | 14 | 14 |

## WEEK THREE - ( 1st August - 7th August ) 1st August, Monday 3ml cultures made for the preparation of competent cells of DH5 alpha cells. 20ml culture prepared for the construct BBa\_K844004. (MaSP without ATG) 2nd August, Tuesday 11 vials of DH5 alpha competent cells were prepared. Plasmid extraction done for the construct BBa\_K844004 (MaSP without ATG). 3rd August, Wednesday As a part of the iGEM 2016 interlab study, transformations were done for the following: Test device1 Test device2 Test device3 Positive control Negative control Also, transformed cells were prepared with the part BBa\_K844008 (MaSP with ATG) 4th August, Thursday Colonies were seen for all six of the transformed plates. The transformation procedure was hence successful. 7th August, Sunday 20ml Culture was prepared for the screening of BBa\_K844008 (MaSP with ATG). Also, 20ml cultures were prepared for each of test device 1, test device 2, and test device 3 for the interlab study.

## WEEK FOUR - ( 8th August - 12th August ) 8th August, Monday PPlasmid extraction performed for the screening of the parts BBa\_K844008 (MaSP with ATG); and test device 1, test device 2, test device 3 for the interlab study. 9th August, Tuesday Gel Electrophoresis with 1% agarose gel was done for BBa\_K844008 (MaSP with ATG), test device 1, test device 2, test device 3 for screening. The run was not successful due to gel over flow. 10th August, Wednesday 20ml Culture was prepared for screening of BBa\_K844008 (MaSP with ATG), test device 1, test device 2, test device 3. 11th August, Thursday Plasmid extraction for the screening of BBa\_K844008 (MaSP with ATG), test device 1, test device 2, test device 3 was done. 12th August, Friday Gel electrophoresis with 1% agarose gel was done for BBa\_K844008 (MaSP with ATG), test device 1, test device 2, test device 3. ![](https://static.igem.org/mediawiki/2016/e/e2/T--IIT_Kharagpur--12_aug.jpg)

## WEEK FIVE - ( 15th August - 18th August ) 16th August, Tuesday Plasmid extraction of BBa\_K844008 (MaSP with ATG), test device 1, test device 2, and test device 3 was done with mini-prep kit. Digestion of above obtained plasmids with E And P was carried out. 17th August, Wednesday 1% agarose gel was prepared for digested plasmids PCR of protease construct for HIV site primers was done PCR parameters: {95 degree 2 minutes [95 degree 30 seconds 60 degrees 30 sec 68 degree 2 min] X35 cycles 68 degree 10 min} PCR products run in 1% agarose gel. Amplification not upto expectation. Repetition of the experiment, with different conditions suggested. Note: Too many non specific products were observed that is why the same was repeated next week. 18th August, Thursday PCR of protease construct for HIV site primers was done. PCR products were run in 1% agarose gel. ![](https://static.igem.org/mediawiki/2016/0/03/T--IIT_Kharagpur--18_aug.jpg) PCR Parameters: {95 degree 2 minutes [95 degree 20 seconds 62 degrees 30 sec 68 degree 2 min 15 sec] X35 cycles 68 degree 10 min} Amplification not upto expectation. Repetition of the experiment, with different conditions suggested.

## WEEK SIX - ( 22nd August - 26th August ) 22th August, Monday PCR for both fret and silk constructs at two conditions was done. ![](https://static.igem.org/mediawiki/2016/9/90/T--IIT_Kharagpur--22_aug.jpg) Condition 1 {95 degree 5 minutes Taq DNA polymerase is added. [95 degree 30 seconds 57.5 degrees 40 sec 68 degree 40 sec] X35 cycles 68 degree 10 min} Condition 2 {95 degree 2 minutes [95 degree 30 seconds 58 degrees 1 min 68 degree 2 min] X35 cycles 68 degree 10 min} 23rd August, Tuesday PCR for FRET construct was done. PCR Parameters: {95 degree 2 minutes [95 degree 30 seconds 57 degrees 40 sec 68 degree 2 min] X35 cycles 68 degree 10 min} 24th August, Wednesday Gel run for FRET construct was performed. ![](https://static.igem.org/mediawiki/2016/e/e0/T--IIT_Kharagpur--24_aug.jpg) 25th August, Thursday PCR for silk construct was done with the following parameters: {95 degree 2 minutes [95 degree 30 seconds 57 degrees 40 sec 68 degree 2 min] X35 cycles 68 degree 10 min} ![](https://static.igem.org/mediawiki/2016/e/ee/T--IIT_Kharagpur--25_aug.jpg) 26th August, Friday PCR for FRET construct was done. PCR Parameters: {95 degree 2 minutes [95 degree 30 seconds 58 degrees 1 min 68 degree 2 min] X35 cycles 68 degree 10 min} ![](https://static.igem.org/mediawiki/2016/3/33/T--IIT_Kharagpur--26_aug.jpg)

## WEEK SEVEN - ( 29th August - 2nd September ) 29th August, Monday Nano drop analysis for pSB1K3( Kanamycin backbone) was done. Result:Obtained concentration was 192.6mg/ul. 31st August, Wednesday Digestion for Kanamycin backbone and FRET PCR with E And P | | K backbone | FRET PCR | | --- | --- | --- | | Component | Volume(ul) | Volume(ul) | | DNA | 6 | 2.3 | | 2.1 Buffer | 2 | 2 | | ECoRI | 1 | 1 | | Psti | 1 | 1 | | Nf H2O | 10 | 13.7 | 1st September, Thursday Ligation for Kanamycin backbone and FRET PCR with E and P was done Vector:Insert=1:3 2nd September, Friday Transformation for Kanamycin backbone and FRET PCR with E and P failed. Note: Reason was determined to be inadequate amount of the PCR product

## WEEK EIGHT - ( 8th September - 10th September ) 8th September, Thursday 2 ml culture of CFP and YFP was prepared. PCR done for FRET construct and silk construct Digestion with E and P of FRET PCR product was done. | | FRET PCR | | --- | --- | | Component | Volume(ul) | | DNA | 19 | | 2.1 Buffer | 2.5 | | ECoRI | 1 | | PstI | 1 | | NF H2O | 1.5 | ![](https://static.igem.org/mediawiki/2016/8/87/T--IIT_Kharagpur--8_sep.jpg) 9th September, Friday PCR purification of digested FRET PCR product was done. Plasmid isolation of YFP and CFP was performed. YFP: 250 ng/ul CFP: 218 ng/ul Glycerol stock for CFP and YFP was prepared. Restriction digestion(overnight) of YFP and cleavage site was done. | | YFP | | --- | --- | | Component | Volume(ul) | | DNA | 5 | | ECoR buffer | 2 | | ECoRI | 1 | | NF H2O | 12 | | | Cleavage Site | | --- | --- | | Component | Volume(ul) | | DNA | 10 | | 3.1 Buffer | 2 | | ECoRI | 1 | | Xbal | 1 | | NF H2O | 6 | 10th September, Saturday PCR purification of YFP digested with E was done. Digestion of purified YFP was done with S for 4 hrs at 37 degrees. | | YFP | | --- | --- | | Component | Volume(ul) | | DNA | 20 | | Cutsmart Buffer | 2.5 | | Spel | 1 | | NF H2O | 1.5 |

## WEEK NINE - ( 12th September - 13th September ) 12th September, Monday 22 ml cultures of CFP, YFP, HIV site, MASP with ATG and MASP without ATG was prepared. 13th September, Tuesday Glycerol stock and plasmid extraction of CFP, YFP, HIV site, MASP with ATG and MASP without ATG was performed. CFP:437.5 ng/ul YFP: 286.7 ng/ul HIV Cleavage Site: 357.1 ng/ul MaSP with ATG: 268.2 ng/ul MaSP w/o ATG: 123ng/ul

## WEEK TEN - ( 19th September - 24th September ) 19th September, Monday PCR for FRET (2 sets) Digestion with E and X for HIV site and E for CFP was successfully done. | | CFP | | --- | --- | | Component | Volume(ul) | | DNA | 20 | | ECoR buffer | 2 | | ECoRI | 1 | | NF H2O | 14.7 | | | Cleavage Site | | --- | --- | | Component | Volume(ul) | | DNA | 2.8 | | 3.1 Buffer | 2 | | ECoRI | 1 | | Xbal | 1 | | NF H2O | 13.2 | 20th September, Tuesday PCR purification of CFP digested with E was done. Digestion with S of purified product was done for 6 hours at 37 degrees. | | CFP | | --- | --- | | Component | Volume(ul) | | DNA | 20 | | Cutsmart Buffer | 2.5 | | Spel | 1 | | NF H2O | 1.5 | Gel extraction of digested CFP and HIV site was successfully performed. The gel pieces stored at 4 degrees ![](https://static.igem.org/mediawiki/2016/4/49/T--IIT_Kharagpur--20_sep.jpg) 21st September, Wednesday Gel extraction of stored gel pieces Amount of extracted DNA too low to proceed to ligation step 22nd September, Thursday S digestion of CFP and X digestion of HIV 23rd September, Friday PCR purification of above digested followed by E digestion of both for 6 hrs at 37 degrees Gel extraction after completion of digestion followed by ligation of E and X digested HIV site and E and S digested CFP at 16 degrees overnight 24th September, Saturday Transformed DH5 alpha cells with ligation mix and plating on A+K plates

## WEEK ELEVEN - ( 25th September - 2nd October ) 25th September, Sunday Colonies observed on incubated plates 26th September, Mondayy 18 colonies from the 1:3 plate were put into 1 ml media overnight for screening 14 colonies from the 1:5 plate were put into 1 ml media overnight for screening Digestion of YFP with X and P Digestion of HIV site with S and P 27th September, Tuesday PCR for FRET (2 sets) Gel extraction for PCR products and digestion with P Digestion of K backbone with P Screening for 18 colonies from the 1:3 plate and 14 colonies from the 1:5 plate Colonies 1, 4, 7, 10 from 1:3 plate showed a shift Colonies 1, 5, 11 from 1:5 plate showed a shift ![](https://static.igem.org/mediawiki/2016/a/aa/T--IIT_Kharagpur--27_sep.jpg) Ligation of YFP digested with X and P with HIV site digested with S and P 28th September, Wednesday PCR purification of P digested K backbone and FRET PCR followed by E digestion Gel Extraction of the digested products followed by ligation Colonies observed of ligated YFP and HIV site observed 29th September, Thursday 25 colonies from the ligated YFP and HIV site plate picked for screening X and P digestion for CFP+ HIV 30th September, Friday Screening of YFP + HIV Gel run X and P digested CFP + HIV 20 ml cultures for plasmid extraction of K-backbone, CFP+HIV and HIV Site, YFP+HIV (colony 7) and YFP+HIV (colony 13) 1st October, Saturday Plasmid extractions for the above cultures Digestion with X and P for YFP+HIV (colony 7) and CFP+HIV Gel run for the digested YFP+HIV (colony 7) and CFP+HIV Digestion with X of YFP+HIV Digestion with S of CFP 2nd October, Sunday PCR purification of the digested products Digestion with E of S digested CFP and X digested YFP+HIV Gel extraction of the digested product Ligation of E and S digested CFP and E and X digested YFP+HIV

## WEEK TWELVE - ( 3rd October - 9th October ) 3rd October, Monday Transformation of the Ligated product onto A+K plates. 4th October, Tuesday Growth was observed on both the plates. 1 ml culture was given for 14 colonies of each of the plates, for the purpose of screening. 5th October, Wednesday 1% Agarose gel was run for the plasmids extracted from the overnight cultures. Sufficient number show the desired shift. 20 ml culture was given for miniprep extraction of CFP+HIV+YFP form colony 10 and 15 of the 1:3 ligation plate 6th October, Thursday PCR was run (2 sets) The PCR product was run on 1% agarose gel for extraction. Amount too low to continue. Miniprep for CFP+HIV+YFP Colony 10: 187.5 ng/ul Colony 15: 185 ng/ul Digestion with X and P for the isolated plasmid (overnight) | | CFP+HIV+YFP | | --- | --- | | Components | Volume(µl) | | DNA | 6 | | 3.1 Buffer | 2 | | Xba1 | 1 | | Pstl | 1 | | NF H2O | 10 | 7th October, Friday 1% agarose gel was run for the digested product. Bands appear as expected 8th October, Saturday 20 ml culture for plasmid extraction of CFP+HIV+YFP (Colony 10) 9th October, Sunday Mini prep extraction of CFP+HIV+YFP CFP+HIV+YFP: 94 ng/µl PCR was done (2 sets) for FRET No amplification was seen

## WEEK THIRTEEN - ( 11th October - 16th October ) 11th October, Tuesday PCR was done (2 sets) for FRET No amplification was seen 13th October, Thursday Digestions with X and P of CFP in pSB1C3 backbone and CFP+HIV, YFP+HIV and CFP+HIV+YFP all in pSB1AK8 backbone. | | CFP+HIV+YFP | CFP+HIV | HIV+YFP | CFP | | --- | --- | --- | --- | --- | | Component |Volume (µl) Volume (µl) | Volume (µl) | Volume (µl) | Volume (µl) | | DNA | 6 | 4 | 6 | 3 | | 3.1 Buffer | 2 | 2 | 2 | 2 | | Xba1 | 1 | 1 | 1 | 1 | | Pstl | 1 | 1 | 1 | 1 | | NF H2O | 10 | 12 | 10 | 13 | 14th October, Friday 1% Agarose gel for extraction of all above. Significant amount observed only in the digested CFP, whereas all the rest proved unsuccessful. 20 ml cultures given for extraction of CFP+HIV, YFP+HIV and CFP+HIV+YFP. 15th October, Saturday Mini prep done for CFP+HIV, YFP+HIV and CFP+HIV+YFP. CFP+HIV: 84 ng/ul HIV+YFP: 92 ng/ul CFP+HIV+YFP: 87 ng/ul X and P digestions set up for all three isolated plasmids | | CFP+HIV+YFP | CFP+HIV | HIV+YFP | | --- | --- | --- | --- | | Components | Volume (µl) | Volume (µl) | Volume (µl) | | DNA | 13 | 13 | 13 | | 3.1 Buffer | 2 | 2 | 2 | | Xbal | 1 | 1 | 1 | | Pstl | 1 | 1 | 1 | | NF H2O | 3 | 3 | 3 | 16th October, Sunday 1% agarose gel run for gel extraction of digested products. Digestion with X and P of CFP and C+H+Y

## WEEK FOURTEEN - ( 17th October - 23rd October ) 17th October, Monday % Agarose gel run for Gel Extraction of digested product. Ligation with ratio 1:3 done using the backbone from CFP and Insert from CFP+HIV+YFP 30 ml culture given for plasmid extraction of CFP+HIV+YFP. 18th October, Tuesday Mini Prep for CFP+HIV+YFP CFP+HIV+YFP: 275 ng/ul X and P digestion of the CFP and CFP+HIV+YFP 1% Agarose Gel for Gel Extraction of the above digested products Quick Ligation was set up for the the digested CFP backbone and CFP+HIV+YFP 1:3 ratio was used Transformation was done on Cam plates Overnight Ligation set up for CFP and CFP+HIV+YFP in the ratio 1:3 Transformation done for the previously set overnight ligation 19th October, Wednesday %No colonies observed on the quick ligated plate Colonies observed on plate whose ligation was done on 17th october 1 ml Cultures for 16 colonies of the plate given overnight Transformation done for the ligated products from 18th October. [![](https://static.igem.org/mediawiki/2016/thumb/b/b1/T--IIT_Kharagpur--kgp_logo.png/799px-T--IIT_Kharagpur--kgp_logo.png)](http://iitkgp.ac.in) [![](https://static.igem.org/mediawiki/2016/thumb/b/bc/T--IIT_Kharagpur--dbt_logo.png/799px-T--IIT_Kharagpur--dbt_logo.png)](http://syntheticbioindia.weebly.com/ibec-2016.html) Connect [![](https://static.igem.org/mediawiki/2016/2/26/T--IIT_Kharagpur--fb.png)](https://www.facebook.com/iGEMIITKharagpur) [![](https://static.igem.org/mediawiki/2016/6/68/T--IIT_Kharagpur--twitter.png)](https://twitter.com/igem_iitkgp) [![](https://static.igem.org/mediawiki/2016/d/d6/T--IIT_Kharagpur--youtube.png)](https://www.youtube.com/channel/UC2NUjcEMwVyNxdLtsti3VRw) Mail Us