G1: cry weapon system

G2: population control system

G3: Low-temperature control system

1

Basic training of iGEM Biobrick assembly
Project discussion and work division

2

3

Preparation of plasmid DNA
(K115002+tetR+PSB1K3)
Using PCR and electrophoresis to check the device

4

5

6

Paper survey for crystal proteins.

7

8

Digestion of the backbone, the aforementioned plasmid DNA, and the promoter J23101.
Ligation of J23101+K115002+tetR+terminator

9

Paper survey and discussion:
a. A plasmid encoding a combination of mosquito-larvicidal genes from Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus confers toxicity against a broad range of mosquito larvae when expressed in Gram-negative bacteria
b. Genetically engineering a 130-kDa Bacillus thuringiensis Cry11Aa Toxin Expressed In E.coli
c. Complete Sequence and Organization of pBtoxis, the Toxin-Coding
Plasmid of Bacillus thuringiensis subsp. israelensis

10

11

Preparation for D’.

R0062 and B0034+ E1010+J61048 plasmid digestion.

Ligation for above two parts.
Electrophoresis to check.
Sequencing for D’.

12

Transformation of J23101+K115002+tetR+terminator

13

Sequencing of J23101+K115002+tetR+terminator

14

Waiting for the sequencing results of J23101+K115002+tetR+terminator

15

16

17

18

19

20

Group discussion and choose the target insect species to terminate.

21

Group discussion

22

Find the species of bacterium in the NCBI data base.

Digestion, Ligation of A+B’+PSB3K3

23

24

Buy the Bacillus we need from BCRC in Hsin-Chu: 15860 (Bacillus thuringiensis subsp. israelensis) and 11029 (Bacillus thuringiensis)

Prepare backbone.

25

26

27

Electrophoresis to check backbone plasmid A,C,K

28

Transformation of pTet+RBS+luxR

Transformation of A+B’+PSB3K3

29

Failed to transform A+B’ to PSB3K3.

30

Transformation of A+B’+PSB1A3

31

1

Digestion of A+B’+PSB1A3.
Transformation of A+B’ from PSB1A3 to PSB3K3.

2

Active Bucillus thuringiensis subsp. Israelensis.
Prepare agar plate for Bti.

Obtained RBS+luxI+terminator from Team_NCTU 2009

3

Preparation of B. toxis

Colony PCR of RBS+luxI+terminator

Colony PCR of A+B’+PSB3K3

4

Preparing plasmid of PRR1 and PRR2

Preparing plasmid of A+B’+PSB3K3

5

Preparing plasmid of B. toxis

Preparing plasmid of RBS+luxI+terminator

Waiting for the sequencing results of A+B’+PSB3K3

6

7

8

9

10

Failed to obtain the genome

Ligation of C

11

Preparing plasmid of B. toxis (with increased lysozyme)

Transform A+B’+PSB3K3 to EPI300

12

Preparation of Flow Cytometry

13

Flow Cytometry practice

14

15

16

Preparing plasmid of B. toxis with different methods

17

Preparation of Flow Cytometry

18

Ligation of Ptet+RBS+luxR

Using Flow Cytometry to obtain the data of A+B’(for 37°C and 30°C)

19

20

21

22

23

Preparation of Flow Cytometry

24

Using Flow Cytometry to obtain the data of A+B’(for 37°C and 30°C)

25

Preparation of Flow Cytometry

26

Using Flow Cytometry to obtain the data of A+B’ (for 40°C, 37°C and 25°C)

27

Extract genomic DNA of Bti. by liquid nitrogen

28

Find the best PCR condition by gradient PCR (taq polymerase kit) and check cry11Aa in the Bti.

Ligation of RR+RT

29

PCR on the best condition by using taq-KOD plus polymerase. (fail: nothing!!)

30

PCR on the best condition by using Blend-taq polymerase.

Preparation of Flow Cytometry

31

Ligation of cry11Aa and TA vector and transform plasmids into DH5α.

Transformation for
RBS+luxR+RBS+luxI+
double ter sites

Using Flow Cytometry to obtain the data of A+B’(for 40°C, 37°C and 25°C)

1

2

Prepare plasmid of cry11A from DH5α to digest. And check DNA fragments size by electrophoresis. (fail)

Digest plasmidRRRIT

3

4

Prepare device C by ligation RBS+luxR and RBS+luxI+ter first and then Ptet.

Sequencing to check
èfail

5

Repeat steps of TA cloning, ligation, transformation and digestion of plasmid by Not1 and EcoR1 restriction enzyme. (success!!)

6

7

Sequencing to check cry11Aa

8

Design primers for PCR:
a. mutagenesis of two enzyme sites(Spe1 & EcoR1) on cry11Aa.
b. EXSP primers

9

10

Receive the sequencing result of cry11Aa (success!!)

Digestion, Ligation of A+B’+PSB1C3 and A+ PSB1C3

11

Transformation of A+B’+PSB1C3 and A+ PSB1C3

12

PCR mutagenesis at two enzyme sites --- EcoR1 and Spe1

13

Colony PCR of A+B’+PSB1C3

14

15

Sequencing of solely Ptet, RBS+luxR,
RBS+luxI+ter sites

èall correct

16

Digest to check the PCR product fragments. (fail: EcoR1 & Spe1 have not removed)

17

Repeat steps of single mutation at EcoR1 site by gradient PCR (KOD plus) to find the best condition.

18

19

20

21

Ligation of PCR fragments and TA vector and transform plasmids into DH5α.

22

Prepare device C by ligation Ptet and RBS+luxR first and then RBS+luxI+ter

Sequencing to check
èfail

23

24

Prepare plasmid of cry11Aa mutated at EcoR1 site.
Digest plasmids to check the cry11Aa fragments whether removed EcoR1 site or not.
(success!!)

25

Repeat steps of single mutation at Spe1 site by PCR (KOD plus)
Ligation, transformation and prepare plasmid.
Digest plasmids to check the cry11Aa fragments whether removed Spe1 site or not.
(fail!!)

26

27

Dilute plasmid (1000X) and repeat PCR mutagenesis at Spe1 site. (fail: nothing but primer dimers on electrophoresis result!!)

28

29

30

Dilute plasmid (1000X, 500X, 1X) and repeat PCR mutagenesis at Spe1 site. (success in 1X!!)

1

Digest and remove mother template DNA with Dpn1.

2

Ligation, transformation and prepare plasmid.
Digest plasmids to check the cry11Aa fragments whether removed Spe1 site or not.
(success!!)

3

Cut mRFP from plasmid with Ptet+ mRFP by digestion

4

Add EXSP sites on ends of cry11Aa (which has been removed the two enzyme sites) by PCR.

5

Ligation of PCR product and psb1C3 backbone. Transform into DH5α.

6

Colony PCR to check the fragments. (fail: only 1.5bp!!)

7

8

Add EXSP sites on ends of cry11Aa by PCR again.
Check fragments size by electrophoresis and purify.

Ligation of Ptet and RBS+luxR

9

Digest EcoR1 and Pst1 site on purified cry11Aa and psb1C3 plasmid. Ligation and transformation

Transform the above

10

Colony PCR to check the fragments. (fail: only 1.5bp!!)
Digest Not1 site on purified cry11Aa (with EXSP) and psb1C3 plasmid.

11

Ligation and transformation

12

Colony PCR to check the fragments. (fail: nothing!!)

Digestion for Ptet+
RBS+luxR

13

Digest Xba1 and Spe1 site on purified cry11Aa and psb1C3 plasmid.

Electrophoresis for checking digestion

14

Ligation and transformation

Digestion for Ptet+
RBS+luxR by different protocol

Digestion, Ligation of J23101+K115002+tetR+terminator +PSB1C3

15

Colony PCR to check the fragments. (fail: only 1.5bp!!)

Colony PCR for Ptet+RBS+luxR

Transformation of J23101+K115002+tetR+terminator+PSB1C3

16

Check PCR product by digestion and electrophoresis. (fail!!)

Colony PCR for Ptet+RBS+luxR from another protocol

17

PCR again with Blend-taq and KOD plus polymerase to obtain cry11Aa (which has been removed the two enzyme sites) from TA vector.

Ligation for device
C, and transform it.

Preparation of plasmid DNA(J23101+K115002+tetR+terminator)

18

Check fragments size.( smear!!)
Change PCR condition and PCR again. (success!!)
Add EXSP site on ends of cry11Aa by gradient PCR.
Check fragments size by electrophoresis. (fail!!)
Design the new primers to add EXSP sites.

Colony PCR for C, pick correct transformed E.coli to incubate

19

Prepare psb1C3 backbone. (Digestion with EX, ES, XS, XP and 5’phosphatase)

Check result by digestion PRRRIT plasmid and electrophoresis

20

Add EXSP sites by PCR with two different protocols and new primers. (success!!)

Sequencing èfail

21

Digestion( by EP sites), ligation and transformation

22

Colony PCR to check the fragments size. (success in NO.3!!!!)

Transfer Ptet+RBS+
luxR to 1C3

23

To check E&S sites of our cry11Aa fragment have been removed in the plasmid, we digest it by EcoR1, Spe1 respectively.(success:4Kb!!)

24

To check the plasmid is not backbone-backbone ligation, we use the colony PCR( by EXSP primer ) to check its size.(success:2Kb!!)

25

!!!!Send our parts to MIT!!!!