The 2016 NEFU iGEM team was recruited at the very beginning of November 2015, just followed the end of the iGEM competition of 2015. After our iGEM team was established, the old guys in our team 2015 give a series of classes to teach every member in our team a foundational knowledge about molecular biology, microbiology and safety. During this period, we have also learned some classic projects of former team which gives us a lot of inspiration on designing our project. The study of fundamental knowledge lasted 3 months.

March to June

We started to design our own project since the middle of March. We put forward three ideas this year, we were divided into three groups to improve the project respectively. We searched and studied lots of papers to build the theoretical framework for each topic during this period.

July to August

We started to learn how to do experiment in our lab in June. In order to avoid lab accident, we all received laboratory safety training before we formally step into the lab. We attended Central China iGEM Consortium (CCiC) at the end of August. During the conference we showed our project and communicated with other teams and gained a lot of inspiration to develop a better system.

September

In this month we started to focus on the group 1’s project. Since the project of group 1 is the most maturity one, we decided to use their project to apply this year’s competition. We had also developed cooperation with NEU_China in this month. Meanwhile we had organized several social activities in this month, in order to expand the influence of iGEM

October

Time flies. We seized the time to make all the experiments done and put great efforts for the wiki building and preparing the presentation for the Giant Jamboree.

Use restriction enzyme to digest the pET-14b backbone. (Vector: pET-14b-9×Z domain)

Using gel electrophoresis to confirm the concentration ratio between the backbone and the insert. (Vector: pET-14b-Pmsp3-Mms13-EGFP)

Enzyme digestion for detection. (vector: pET-14b-Pmsp3-Mms13-EGFP)

Digestion of intein. (Vector: pET-14b-Amilcp-intein-Spytag)

Using gel electrophoresis to confirm the concentration ratio between the backbone(pET-14b) and the insert. (Vector: Amilcp: Intein=2:20:1) (Vector: pET-14b-Amilcp-intein-Spytag)

Enzyme digestion (9x Z domain) (vector: pET-14b-9×Z domain)

BamH I	Kpn I	10x CutSmart Buffer	plasmid	Temperature	Time
1µl	1µl	3µl	25µl	37°C	4h

Enzyme digestion (pET14b plasmid) (Vector: pET-14b-9×Z domain)

KpnI	BamHI	10x CutSmart Buffer	plasmid	ddH2O	Temperature	Time
1µl	1µl	3µl	2µl	23µl	37°C	4h

Enzyme digestion (Vector: pET-14b-Pmsp3-EGFP)

EGFP	KpnI	NotI	10*smart buffer	H2O
25µl	1µl	1µl	3µl	0µl

Enzyme digestion (Vector: pET-14b-Pmsp3-EGFP)

Pet14b-Pmsp3	KpnI	NotI	10*smart buffer	H2O
5µl	1µl	1µl	3µl	20µl

Ligation (Vector: pET-14b-Pmsp3-EGFP)

pet14b-pmsp3	10*buffer	T4 Ligase	H2O	Insert: EGFP
1µl	1.5µl	0.5µl	11.2µl	0.8µl

E. coli transformed with vector (pET-14b-Pmsp3-EGFP) cultured on LB-ampicillin medium for 14h.

EGFP

Primers	Primer F	CGGGTACCGTGAGCAAGGGCGAGGA
Primers	Primer R	ATAAGAATGCGGCCGCTTACTTGTACAGCTCGTCCATG
PCR system (50µl)		Parameters
PCR system (50µl)		Procedure	Temperature	Time
2×Taq Master Mix	25µl	Predenature	94℃	5min
PrimerF (10µM)	1 µl	Denature	94℃	30sec
PrimerR (10µM)	1 µl	Annealing	55℃	30sec
ddH2O	22µl	Extension	72℃	60sec
Template	1µl	Final Elongation	72℃	10min
dNTPs	Included in premix	Final Hold	4℃	∞
Buffer	Included in premix	Cycle	30 cycles

Mms13

Primers	Primer F	GGGGTACCCCCTTTCACCTTGCCCC
Primers	Primer R	CCGCTCGAGGGCCAGTTCGTCCCGC
PCR system (50µl)		Parameters
PCR system (50µl)		Procedure	Temperature	Time
2×Taq Master Mix	25µl	Predenature	94℃	5min
PrimerF (10µM)	1 µl	Denature	94℃	30sec
PrimerR (10µM)	1 µl	Annealing	60℃	30sec
ddH2O	22µl	Extension	72℃	45sec
Template	1µl	Final Elongation	72℃	10min
dNTPs	Included in premix	Final Hold	4℃	∞
Buffer	Included in premix	Cycle	30 cycles

pmsp3（488bp）

Primers	Primer F	GAAGATCTACTAGTACGTTGAATCCCAGCG
Primers	Primer P	GCTCTAGAGGGAAACCGTTGTGGTCTCATAGAAAGGCTCCCTCATAG
PCR system (50µl)		Parameters
PCR system (50µl)		Procedure	Temperature	Time
2×Taq Master Mix	25µl	Predenature	94℃	5min
PrimerF (10µM)	1 µl	Denature	94℃	30sec
PrimerR (10µM)	1 µl	Annealing	55℃	30sec
ddH2O	22µl	Extension	72℃	30sec
Template	1µl	Final Elongation	72℃	10min
dNTPs	Included in premix	Final Hold	4℃	∞
Buffer	Included in premix	Cycle	30 cycles

Colony PCR (pmsp3-1 pmsp3-2)

Primers	Primer F	AGCCCGATCTTCCCCATC
Primers	Primer P	TTCAGCAAAAAACCCCTCAA
PCR system (10µl)		Parameters
PCR system (10µl)		Procedure	Temperature	Time
10×Dream Taq Buffer	1µl	Predenature	94℃	5min
PrimerF (100µM)	0.05 µl	Denature	94℃	30sec
PrimerR (100µM)	0.05 µl	Annealing	58℃	30sec
ddH2O	8.4µl	Extension	72℃	30sec
Template: Pick a single colony and dip in H2O		Final Elongation	72℃	10min
dNTPs	0.2	Final Hold	4℃	∞
Taq Enzyme	0.05	Cycle	30 cycles

amilcp

Primers	Primer F	CGGAATTCAGTGTGATCGCTAAACAAATGAC
Primers	Primer P	CGGGATCCTTATTAGGCGACCACAGGTT
PCR system (50µl)		Parameters
PCR system (50µl)		Procedure	Temperature	Time
2×Taq Master Mix	25µl	Predenature	94℃	5min
PrimerF (10µM)	1 µl	Denature	94℃	30sec
PrimerR (10µM)	1 µl	Annealing	50℃	40sec
ddH2O	22µl	Extension	72℃	30sec
Template	1µl	Final Elongation	72℃	10min
dNTPs	Included in premix	Final Hold	4℃	∞
Buffer	Included in premix	Cycle	30 cycles

EGFP+Pmsp3

Primers	Primer F	CGGGTACCGTGAGCAAGGGCGAGGA
Primers	Primer P	ATAAGAATGCGGCCGCTTACTTGTACAGCTCGTCCATG
PCR system (25µl)		Parameters
PCR system (25µl)		Procedure	Temperature	Time
2×Taq Master Mix	12.5µl	Predenature	94℃	5min
PrimerF (10µM)	1 µl	Denature	94℃	30sec
PrimerR (10µM)	1 µl	Annealing	58℃	30sec
ddH2O	9.5µl	Extension	72℃	60sec
Template	1µl	Final Elongation	72℃	10min
dNTPs	Included in premix	Final Hold	4℃	∞
Buffer	Included in premix	Cycle	30 cycles

Colony PCR (EGFP+Pmsp3)

Primers	Primer F	AGCCCGATCTTCCCCATC
Primers	Primer P	TTCAGCAAAAAACCCCTCAA
PCR system (10µl)		Parameters
PCR system (10µl)		Procedure	Temperature	Time
10×Dream Taq Buffer	1µl	Predenature	94℃	5min
PrimerF (100µM)	0.05 µl	Denature	94℃	30sec
PrimerR (100µM)	0.05 µl	Annealing	58℃	30sec
ddH2O	8.4µl	Extension	72℃	30sec
Template: Pick a single colony and dip in H2O		Final Elongation	72℃	10min
dNTPs	0.2	Final Hold	4℃	∞
Taq Enzyme	0.05	Cycle	30 cycles

16S rRNA Gene cloning

1.Each sample of bacteria (1-5ml) was in a tube. Centrifugation was carried out at 12,000 rpm for 1 min at 4°C. The supernatant was discarded, and the pellet was processed for each procedure as follows.

2. Add 180μL Buffer GTL to the pellet to suspend.

3. 20μL Proteinase K was added to the tube and vortex. Then the samples were incubated at 56°C for 30 min until the solution is clear.

4. Add 20μL Buffer GL. The mixture was then briefly mixed on a vortex mixer and centrifuged at 10,000 g for 5 min. Then add 200μL anhydrous ethanol and still vortex.

5. Add the solution obtained from step 4 (include the pellet) to the adsorption column with collection tube, centrifuge at 12000rpm for 1 min, discard the effluent. Adsorption column was put back in the collection tube.

6. Add 500μL Buffer GW1 to the adsorption column, centrifuge at 12000rpm for 1 min. Discard the effluent. Adsorption column was put back in the collection tube.

7. Add 500μL Buffer GW2 to the adsorption column, centrifuge at 12000rpm for 1 min. Discard the effluent. Adsorption column was put back in the collection tube.

8. Centrifuge at 12,000rpm for 2 min, discard the effluent. Put the adsorption column at room temperature until dry out.

9. Place an adsorption column in a new tube, add the 50μLBuffer GE to the column. Put at room temperature for 2-5 min. Centrifuge at 12,000rpm for 1 min. Collect DNA solution, store at -20°C.

Make competence AMB-1 and transformation

Growth

1.Add 1ml bacterial solution to 100ml LB media.

2.Grow at 30°C, 200rpm

3.after 3 and half hours, the OD600 is 0.44

4.after 5 hours, OD600 is 0.85

Make competence cells

1.200ml bacterial solution, stored in ice for 10 min.

2.Centrifuged at 8000 rpm for 30 min at 4℃ .

3.Wash bacteria twice with 272mmol sucrose 10mM TES (PH 7.5). Centrifuged at 3000 rpm for 12 min

4.Suspend in 2ml TES, put in 1.5ml tube, stored in -40℃

Electroporation

Bacteria cultured on the MSGM media. Single colony was spotted.

Fig. 1 AMB-1 cultured on the MSGM media. Single colony was spotted.

Fig. 2 AMB-1 cultured on the LB media. Single colony was spotted.

Team:NEFU China/Notebook