iGEM 2011 — Team ZJU-China

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# Team:ZJU-China/Notebook

### From 2011.igem.org

Notebook - July

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#### Lab Notes

[July](https://2011.igem.org/Team%3AZJU-China/Notebook.html) [August](https://2011.igem.org/Team%3AZJU-China/August.html) [September](https://2011.igem.org/Team%3AZJU-China/September.html)
[October](https://2011.igem.org/Team%3AZJU-China/October.html)

#### Protocol

[Protocol](https://2011.igem.org/Team%3AZJU-China/Protocol.html)

#### Brainstorm

[Brainstorm](https://2011.igem.org/Team%3AZJU-China/Brainstorm.html)

# Lab Notes - July

# Biobrick Group

# Week1

|  |  |
| --- | --- |
| **Day** | **Note** |
| Jul.4th Monday | ▪ Aerobic cultivation of DH5α | ▪ Preparation of apparatus for the formation of biofilm | |
| Jul 5th Tuesday |  |
| Jul.6th Wednesday | |  | | --- | | ▪ Receiving primers ordered previously | |
| Jul.7th Thursday | |  |  | | --- | --- | | ▪ Preparation of the aliquot of the primers | ▪ Something wrong with a shaking incubator | |
| Jul.8th Friday | |  | | --- | | ▪ Preparation of culture plates for the transformations | |
| Jul.9th Saturday | |  |  | | --- | --- | | ▪ Preparation of culture plates for the transformations | ▪ protocols of transformation | |
| Jul.10th Sunday | |  | | --- | | ▪ Several colonies were picked up and cultivated in 5mL LB medium. ▪ryosectioning of biofilm | |

# Week2

|  |  |
| --- | --- |
| **Day** | **Note** |
| Jul.11th Monday | ▪ Set up new LB culture plates with ampicillin and kanamycin | ▪ Sterilization of Glycerol and   Preparation of 25mg/mL kanamycin | ▪Transformation of the parts mentioned on Jul.9th for the second time | ▪Observation the sections | |
| Jul 12th Tuesday | ▪ Pick two colonies of each parts and cultivate them in LB medium | ▪ Transformation of three parts(20J,20H.22B from Kit plates of 2011 Distribution ) which are related to the degradation of Cellulose | ▪Min prep to isolate 10I,12I and 22M | ▪Conservation of 10I,12I,22M and 11P | |
| Jul.13th Wednesday | |  |  |  |  |  | | --- | --- | --- | --- | --- | | ▪ Place the culture plate of 20J,20H and 22B in the fridge. | ▪min prep to isolate 13K ▪onservation of 13K | ▪estriction digest of the parts(12I,10I,22M,13K) with EcoRI and PstI | ▪Gel electrophoresis to analyse restriction fragments | ▪Test the Tm of Primers CP1&CS,NP&NS with 13K. The result of Gel electrophoresis shows that 60.2℃ is the Tm of NS and NP, and 57.4℃ is the Tm of CS and CP1. | |
| Jul.14th Thursday | |  | | --- | | ▪ Test the Tm of the YFP,RFP,tetR,Vgb and nirB primers. The result of Gel electrophoresis shows that the Tm for the primers of Vgb is 54℃ and the Tm for the primers of nirB is 55℃ The RCR of YFP,RFP and tetR failed. | |
| Jul.15th Friday | |  |  |  | | --- | --- | --- | | ▪ PCR(Phusion)  ▪ Digest 10I, 22M, 13K ▪ Used wrong cutter, digestion again. | ▪ Run the results of PCR and the first digestion. The annealing temperature of YFP needed change. The digestion results confirmed | ▪ Run the digestion results of second time. The bands are confirmed. | |
| Jul.16th Saturday | |  |  |  |  |  | | --- | --- | --- | --- | --- | | ▪ Cut the linearized pSB1k3 with E+P | ▪ Purify the digestion results of 22M, 13K, 10I | ▪ Confirm digestion of pSB1k3 by electrophoresis, then purification | ▪ Test Tm of YFP  ▪ ligation: 22M+10I, 13K+10I | ▪ Tm of YFP is 54 degree ▪ transform the ligation results. | |
| Jul.17th Sunday | |  |  |  | | --- | --- | --- | | ▪ PCR 22M  ▪ purify Vgb, RFP, RFP-tetR, YFP-tetR. Gibson PCR. | ▪ ligation the purified the fragments in yesterday. | ▪ 22M PCR  ▪ Transform the ligation results. | |

# Week3

|  |  |
| --- | --- |
| **Day** | **Note** |
| Jul.18th Monday | ▪ Genome extraction of E.coli | ▪ PCR of NirB  ▪ Cut 10I with X+P, cut 22M, 13K with E+S, cut pSB1k3 with E+P | ▪ Ligate: 10I+13K, 10I+22M   ▪ Repeat NirB PCR | ▪ Culture 11P  ▪ Miniprep 10I, 22M, 13K | |
| Jul 19th Tuesday | ▪ mini-prep: 10I+13K, 10I+22M | ▪Insert 10I+13K, 10I+22M into pSB1k3 | ▪Transform: 5E, 3C, 7C, 1K, 1I from the distribution plate | ▪Transform 10I+22M, 10I+13K | |
| Jul.20th Wednesday | |  |  |  |  | | --- | --- | --- | --- | | ▪Colonies of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I confirmed | ▪ Gibson PCR | ▪Colony PCR 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I | ▪PCR NirB | |
| Jul.21th Thursday | |  |  |  | | --- | --- | --- | | ▪PCR verification of 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I | ▪ Gibson PCR ▪ Run the results of PCR verification. Bands confirmed. | ▪ Culture the 22M+10I, 13K+10I, 5E, 3C, 7C, 1K, 1I ▪ Purification of Gibson PCR results | |
| Jul.22th Friday | |  |  |  |  | | --- | --- | --- | --- | | ▪ PCR amplification of Gibson assembly results | ▪ Mini prep: 22M+10I, 22.Presever in -20  ▪ Mini prep: 1K,1I,3C,5E,7C  ▪ Gibson Assembly fail. | ▪ PCR NirB by Phusion  ▪Repeat PCR by changing Pnibr to Gnirbr  ▪Cut the mini and medi prep results with E | ▪ Run the results of PCR and digestion. Fail in PCR of NirB, succeed in 10I+3K and 10I+22M ligation. | |
| Jul.23th Saturday | |  |  |  | | --- | --- | --- | | ▪Double digestion of 13K+10I, 22M+10I, pSB1c3 | ▪ Fail in purification of Medi prep | ▪ PCR NirB  ▪ Ligate NirB+13K+10I, Vgb+22M+10I | |
| Jul.24th Sunday | |  |  |  | | --- | --- | --- | | ▪ Gibson assembly: NirB+RFP+tetR  ▪ Cut results of Gibson assembly and pSB1c3. | ▪ Purify the ligation results in yesterday | ▪ ligate with backbone ▪ Culture 1I, 1K, 3C, 5E, 7C, 11P | |

# Week4

|  |  |
| --- | --- |
| **Day** | **Note** |
| Jul.25th Monday | ▪ Transform Pnirb+13k+10I+pSBK3-2 | ▪ Place the culture in 4 degree | ▪ Medi-and mini-prep of five cultre | ▪Gibson Assembly confirmation by gel | |
| Jul. 26th Tuesday | ▪ Cut Pnirb, Pvgb,(E+S)  ▪ Cut 10I+13K, 10I+22M.(X+P) | ▪ Ligation: vgb+10I+22M, nirB+10I+13K, | ▪ Ligation: pSBK13+vgb+10I+22M, pSBK13+nirB+10I+13K | ▪ Transform the ligation results. | |
| Jul.27th Wednesday | |  |  |  | | --- | --- | --- | | ▪ No colony on the plate  ▪ Gibson assembly | ▪ Run the results of Gibson assembly | ▪ Excise the bands, the purify the DNA  ▪ Gibson Assenmly | |
| Jul.28th Thursday | |  | | --- | | ▪ 2011 China Meet-up @ Hefei | |
| Jul.29th Friday | |  | | --- | | ▪ 2011 China Meet-up @ Hefei | |
| Jul.30th Saturday | | |  | |
| Jul.31th Sunday | |  |  | | --- | --- | | ▪ Culture 22M+10I, 13K+10I | ▪ Cut a0H, 20J, 22B | |

# Data/Protocol

# Jul.13th Wednesday

**Systems of restriction digestion with EcoRI and
PstI**

|  |  |
| --- | --- |
| Plasmid | 1μL (>100ng) |
| EcoRI | 1μL |
| PstI | 1μL |
| 10×Buffer Tango | 2μL |
| ddH2O | 15μL |

![](https://static.igem.org/mediawiki/2011/c/c3/0713program.png)

**Temperature grad**
56℃ 57.4℃ 60.2℃ 62.9℃ 64.3℃ 65.8℃ /p>

**PCR system (test the Tm of the primers
CP1&CS, NP&NS)**

|  |  |
| --- | --- |
| 10×Buffer | 2μL |
| dNTPs | 0.5μL |
| primers CP1 (NP) | 1μL |
| primers CS (NS) | 1μL |
| Template | 1μL |
| rTaq | 0.2μL |
| H2O | 14.5μL |
| Total | 20μL |

Within each compartment are **components**: include
different types of biomass ,substrates , products. biomass is often
divided into active microbial species, inert cells, and extracellular
polymeric substances(EPS).

The components can undergo transformation, transport, and
transfer **processes**. For example, substrate is consumed,
and this leads to the synthesis of new active biomass.

All process affecting each component in each compartment are
mathematically linked together into a **mass balance
equation** that contains rate terms and parameters for each process.

**Model Selection:**Many kinds of Mathematics models
have been founded to describe a system of biofilm. Models of different
dimensions (1d, 2d, 3d) focus on different properties of a biofilm.
Since we care most about the oxygen concentration gradients
perpendicular to the substratum, **numerical
1-dimensional dynamic model(N1)** would be a proper choice for us.

![](http://ung.igem.org/wiki/images/4/42/Biofilmcomp.png)

# Early July

Pre-experiments with biofilm formation with circular and non
circular silicone tube, 24 well plate on different support including
glass, paper, plastic film, rubber. The final material are used based on
the easiness biofilm form on them and on the easiness to observe under
microscope.

![](https://static.igem.org/mediawiki/2011/b/be/20110711-3nP4100X0877.jpg)

# 28th July

13.30: DH5α 11p 5mlX4
23.00: silicone tube set at 37℃ /p>

# 29th July

13.00: LB culture 50ml with circular culture medium. LB culture
50ml with noncircular culture medium.

# 31st July

13.00: -80℃ storing silicone tube. A thick white and loosely bond
substance is seen on the inner wall of the 5mm silicone tube, and on the
inner wall of the 1mm silicone tube a flatter and smoother white
substance. Especially obvious where the tube turns. Possibly because the
speed of culture flow is slower.

Biobrick Group|Biofilm Group

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