iGEM 2016 — Team XJTLU-CHINA

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# Team:XJTLU-CHINA/Notebook

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XJTLU CHINA

* [Home](https://2016.igem.org/Team%3AXJTLU-CHINA)
* Team
  + [Members](https://2016.igem.org/Team%3AXJTLU-CHINA/Team)
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  + [Collaborations](https://2016.igem.org/Team%3AXJTLU-CHINA/Collaborations)
* Project
  + [Project Description](https://2016.igem.org/Team%3AXJTLU-CHINA/Design)
  + [Experiments](https://2016.igem.org/Team%3AXJTLU-CHINA/Experiments)
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* Parts
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  + [Registry Parts Improvement](https://2016.igem.org/Team%3AXJTLU-CHINA/Description)
* Human Practices
  + [Overview](https://2016.igem.org/Team%3AXJTLU-CHINA/HP/overview)
  + [Silver](https://2016.igem.org/Team%3AXJTLU-CHINA/HP/Silver)
  + [Gold](https://2016.igem.org/Team%3AXJTLU-CHINA/HP/Gold)
  + [Public Engagement](https://2016.igem.org/Team%3AXJTLU-CHINA/Engagement)
* [Safety](https://2016.igem.org/Team%3AXJTLU-CHINA/Safety)

# Frequently used Protocols

[Agrose Gel
Electrophoresis](https://static.igem.org/mediawiki/2016/8/81/Protocol-Agrose_Gel_electrophoresisXJTLU16.pdf)
[DNA Gel Extraction
 Work Flow](https://static.igem.org/mediawiki/2016/0/00/Protocol-DNA_Gel_extraction_Work_FlowXJTLU16.pdf)
[Gene Synthesis](https://static.igem.org/mediawiki/2016/d/df/Protocol-Gene_synthesisXJTLU16.pdf)
[Gibson Assembly](https://static.igem.org/mediawiki/2016/d/d2/Protocol-Gibson_assemblyXJTLU16.pdf)

[PCR Amplification
 of DNA Fragment from
 Plasmid](https://static.igem.org/mediawiki/2016/b/b7/Protocol-PCR_amplification_of_DNA_fragment_from_plasmidXJTLU16.pdf)
[Transformation](https://static.igem.org/mediawiki/2016/6/67/Protocol-TransformationXJTLU16.pdf)
[SDS-PAGE](https://static.igem.org/mediawiki/2016/9/95/Protocol-sdspage-XJTLU16.pdf)
[RT-PCR](https://static.igem.org/mediawiki/2016/5/54/RT-PCR_XJTLU16.pdf)

[Fluorescence
measurement](https://static.igem.org/mediawiki/2016/8/83/Fluorescence_measurement-XJTLU16.pdf)
[Colony PCR](https://static.igem.org/mediawiki/2016/a/a7/Colony_PCR-XJTLU16.pdf)
[RNA extraction](https://static.igem.org/mediawiki/2016/8/8d/RNA_extraction-xjtlu16.pdf)
[Gel extraction](https://static.igem.org/mediawiki/2016/a/a2/Gel_extraction-XJTLU16.pdf)

## March:

Overview:

Fragments construction.

3.14

Group 1:

Test of PCR standard for tsf & tufA gene

3.15

Group 1:

Gel electrophoresis of tsf gene and tufA gene

Gel extraction of tsf

TfuA gene PCR (test the optimal condition

Gel electrophoresis of tufA gene

3.17

Group 1:

Plasmid extraction

Isolation of plasmid pACYCDuet from Top.10 cells

3.23

Group 1:

Extraction of QBeta genome

3.26

Group 1:

Transformation of pQBeta7 to competent cell Top.10

3.27

Group 1:

Incubate transformed competent cells

## April:

Overview:

Fragments construction.

Ligation of genome fragments.

4.19

Group 1: Tu PCR.

4.20

Group 1: Tu PCR.

Group 3: gel extraction.

4.25

Group 1: do the pcr purification for Tu gene

4.26

Group 1: Gel extraction.

Group 3: ligate genome fragment 1 and 2.

![](https://static.igem.org/mediawiki/2016/d/d5/4_26.jpeg)

Group 3: Gel extraction.

4.28

Group 1: Rep PCR.

## June:

Overview:

Recombination of plasmids.

6.23

Group 4:

Transformation for QBeta 7
Select HB107 with PUC19 plasmids and cultivation
Result: no rep has been detected.
Construction of pACYC-TS

1. PCR of tsf
Result: tsf has been coamplified
2. Electrophoresis to examine the length of tsf
Result: tsf has correct (expected length)
3. PCR purification
4. pACYC and tsf double digestion with NcoI and BamHI
5. Ligation of pACYC(digested and tsf (digested
Result: the mixture might contain pACYC-ts
6. Transformation of pACYC-ts

6.25

Goup 1:

Check the fragment inserted in pACYC
1. Cell amplification
2. PCR of a fragment on pACYC containing ts
3. Gel electrophoresis
4. Second turn PCR

6.26

Group 1:

Tuf PCR
1. Tu nesting PCR
2. Gel electrophoresis of the PCR product
3. Tu nesting PCR product purification
4. Tu PCR
5. Tel electrophoresis of the PCR product

6.27

Group 4:

HB101 selection and cultivation
1. Select HB101 with PUC19 plasmids and cultivation
2. PCR for testing the product of HB101
3. Select A1¬，B2, C3, D1, E3, F2¬ tubes which contain HB101 with pUC19 into conical flask
4. Cultivate over night

6.28

Group 1:

Ligation of Tu and Transformation of pACYC-tu
1. Double digestion of tu and pACYC
2. Ligation of tu onto pACYC
3. Transformation

6.29

Group 1:

Check whether tu has been inserted into pACYC
1. Cell amplification
2. Tu testing PCR
3. Gel electrophoresis

6.30

Group 1:

Ligation of tu & transformation of pACYC-tu
1. Ligation of tu onto pACYC
2. Transformation

## July:

Overview:

Fragments PCR.

Recombination of plasmids.

Ligation of genome fragments.

7.2

Group 1:

Digestion of plasmid pACYC-DuetI and DNA tufa & ligation and transformation of pACYC-tu

1. double digestion of tu & pACYC

2. Clean up of digested pACYC-DuteI and tufa

3. Ligation of pACYC-DuetI with tfuA

4. Transformation of pACYC-tu into Top-10 cells

![](https://static.igem.org/mediawiki/2016/4/43/7m2d.png)

Group 4:

Transformation fot Duet (Duet to BL21

Check whether tfuA is inserted into pACYC-DuetI

1. Cell amplification

2. Tu testing PCR

3. Gel electrophoresis
Results: none of the colonies selected contain s the recombinant plasmid pACYC-tu

7.5

Group 1:

Amplification of E.coli cells containing pACYC-ts

Group 4:

Double transformation for duet and acyc

7.6

Group 1:

Digest pETDuet-1 with enzymes and test

Gibson assembly of intron a, b and kan

Run electrophoresis to test

Clean up

7.7

Group 1:

Digestion of plasmid pACYC-DyetI and DNA tufa & ligation and transformation of pACYC-tu

1. Digestion and ligation of pACYC-DuetI and tufa (same as the recording on 7.2

Transformation of recombinant plasmids into Top.10 cells

Group 4:

Part II

1. Digest Duet with enzyme

2. Clean up

3. Gibson assembly

4.Transformation to BL21

7.8

Group 1:

PCR testing of tu insert

1. Cell amplification

2. Tu testing PCR (same as the recording on 7.4

3. Gel electrophoresis

Results: A6 has the target length of band

4. Amplification

Result: 600nm (Abs: 0.103

Group 4:

Part II

1. Gibson assembly: intron+ kana

2. Run gel electrophoresis to test

Result: intron a\_ kan was not connected

Intron b+ kan connected a little

3. PCR the bacteria BL21 contain recombinant Duet-1 (Target+ Ndel & Xhot

7.9

Group 1:

Mini prep and planting of pACYC-tu (check pACYC-tu is the only plasmid

1. Planting of transformed Top.10 cells

2. Miniprep of plasmids

Re-testing by PCR of plasmid pACYC-tu (check whether pACYC-tu is the only plasmid

1. PCR testing of ty insert

2. Gel electrophoresis

Results: there is the target insert

Transformation of pACYC-tu into Top.10 E.coli cells

7.10

Group 1:

Amplification of Top.10 E.coli cells containing pACYC-tu

7.11

Group 4:

PCR testing of pACYC

Gel electrophoresis

Digest Duet-1 with Xbal &AfI III

Clean up

Gibson assembly

Transformation Gibson assembly product into BL21

7.12

Group 4:

PCR the bacteria BI21 contain pACYC, intron a-kan-intron b-DuetL(group 1

7.14

Group 4:

Digest Duet-1 with Xbal and Afl II

Clean up

Gel electrophoresis

Gibson assembly

Transformation

7.15

Group 4:

PCR the bacteria Top.10 contain intron a-kan-intron b+-Duet-1(Xbal & afl II

7.25

Group 1:

Construction of pACYC-tu-ts

1. Double digestion with NcoK and BamHI

2. Ligation of tsf onto pACYC-tu

3. PCR for tsf amplification from pACYC-ts

Result: tsf has been amplified successfully

Group 2:

Use bigson assembly ligation genome 1, 2, 3, 4

PCR to replicase genome 1, 2, 3, 4 (S15 system

Group 3:

Digest Duetl WITH Xbal and afl III

Gel electrophoresis

Clean up

Gibson assembly

Clean up

PCR reaction of a+b+k

Gel electrophoresis

Clean up a+b+k

Assemble a+b+k+Duet-1 with Gibson assembly kid

Clean up a+b+k+Duet-1

Transform plasmids (step 9 into Top.10, then plate them onto AMP plates

7.26

Group 1:

Construction of pACYC-tu-ts

1. Double digestion of the pACYC-tu with NcoL and BamHI

2. Ligation of tsf onto pACYC-tu

Group 2:

Amplify the Gibson product (1-2-3-4

PCR

Gel electrophoresis

Bacterial glycerol stock

Extract plasmids

Group 4:

Gibson assembly:

Clean up:

PCR a+k

Pick up colonies from two AMP plates made on 7.24

Inculbate 35 colonies under 37℃ over 2 hours

PCR

Gel electrophoresis

7.27

Group 1:

Ligation & transformation

1. Continue the process of ligation after over night incubation at 16℃

2. Transformation of pACYC-tu-ts

Group 2:

PCR genome 1~4 from plasmid, which was entracted from Top.10

Gel electrophoresis

Results: sample1 and 4 has similar gel electrophoresis result

Group 4:

Get the PCR product +(a+k

Agarose gel electrophoresing of maturase

Gel electrophoresis

Gel electrophoresis

Clean up

7.28

Group 1:

Check the inserted fragment of tsf in pACYC-tu-ts

1. Cell amplification

2. PCR to check the fragment on pACYC-tu-ts to see whether ts can be generated with taq enzyme. 30 tubes were used

3. Gel electrophoresis to see the results from PCR tsf amplification

Result: no target length of the fragments appeared. Experiment need to be repeated.

7.29

Group 1:

Check the inserted fragment of tsf in pACYC-tu-ts (the same procedure as in the experiment on 7.28

Result: still no target length of the fragments appeared

Group 4:

PCR testing of pACYC

Gel electrophoresis

Clean up and nanodrop

PCR the plasmids from bacteria Top.10 contain a-kan-b-Deut-1: D2, B6

PCR testing of pACYC

## August:

Overview:

Recombination of plasmids.

Ligation of genome fragments.

8.1

Group 1:

Check the insert of tsf in pACYC-tu (same as 7.28)
Excluding gel electrophoresis

Group 4:

- PCR testing of pACYC
  Gel electrophoresis
  Clean up+ nanodrop

Gibson assembly

PCR the reaction of a+k (Q5

Gel electrophoresis+ cleanup

PCR the reaction of a+k (S15

8.2

Group 4:

Extract the gel containing a+k (S15)

Gel electrophoresis

Gel extraction

Check the pACYC+ matuase in bacteria

Cell amplification

PCR reaction for each colonies

Gel electrophoresis

PCR: knock our for plasmid in acyc

8.3

Group 4:

Gel electrophoresis of the knock our PCR product

PCR reaction for intron a and kan

Gel electrophoresis

Gibson assembly of intron a and kan

Group 1:

Plasmids (pACYC-ts-tu)

Result: concentration of nucleic acid: 168.1ng/Μl
260/280: 1.92

8.4

Group 1:

Gel electrophoresis of rep fragment from PCR

PCR purification: use QIA quick PCR purification kid (250

Double digestion of Rep and PET-Duet-1

Incubate at 37℃ waterbath for 2 hours

Use MiniElute reaction ceanup kid (QIAGEN Cat No.28204

Ligation of pETDuet-I and Rep

Incubate at 16℃ incubator for overnight

Group 4:

PCR reaction for intron b. (412

Gel electrophoresis

Clean up

Check mutuase in bacteria

Cell amplification

PCR for each colonies

Gel electrophoresis

PCR reaction for intron b

8.5

Group 4:

Gel electrophoresis

PCR of matuase

Transformation of ligation mixture (over night at 16 degrees）

![](https://static.igem.org/mediawiki/2016/3/35/8_5.tiff)

8.8

Group 4:

Geo eoectrophoresis

Knock out T7 promotor of pACYC

Gel electrophoresis

Clean up

8.9

Group 1:

construction of pACYC-tu-ts and transformation of the recombinant plasmid into the competent cells

1. Double digestion of pACYC-tu and tsf

2. Ligation of tsf onto pACYC-tu

3. Transformation

Group 4:

Transformation of pACYC-T7 and maturase into Top.10

Gel electrophoresis

Clean up

8.10

Group 1:

Check whether ts has been inserted in pACYC-tu

Cell amplification

PCR with the amplified cells as template

Construction of pETDuet-Rep

Transformation of pETDuet into Top.10

Group 4:

Gel electrophoresis of PCR products: K1, K2, A1, A2

Clean up

Gel electrophoresis

Digest Duet-1-target with Xbal and Afl III

PCR: 8.10 PCR of intron A

8.11 PCR of kan sample

PCR of intron b

Knock out T7 promotor of pACYC

Gel electrophoresis clean up and nanodrop

8.11

Group 4:

Gibson assembly

Transformation of M-pACYC-T7 into Top.10

Digest Duet-1-target with Afl III and Xbal

Gibson assembly

PCR of a+b+k

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