iGEM 2009 — Team Illinois

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# Team:Illinois/Notebook

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|  |
| --- |
| Contents   * [1 Notebook](#Notebook) * [2 Browse by Summer Lab Team](#Browse_by_Summer_Lab_Team) * [3 Post-Summer Progress](#Post-Summer_Progress) * [4 September 22](#September_22) * [5 September 24](#September_24) * [6 September 25](#September_25) * [7 September 26](#September_26) * [8 September 27](#September_27) * [9 September 28](#September_28) * [10 September 29](#September_29) * [11 September 30](#September_30) * [12 October 1](#October_1) * [13 October 2](#October_2) * [14 October 3](#October_3) * [15 October 4](#October_4) * [16 October 5](#October_5) * [17 October 6](#October_6) * [18 October 7](#October_7) * [19 October 17](#October_17) |

## **Notebook**

This page will include periodic entries during our research taken from our lab notebooks. We will be documenting the progression of our project, any problems or obstacles encountered, and solutions to problems.

## **Browse by Summer Lab Team**

Click on a group name below to view notebook entries and information pertaining to one of our individual summer lab teams.

* [sRNA Library](https://2009.igem.org/Team%3AIllinois/sRNA_Library)
* [Hybrid Promoter](https://2009.igem.org/Team%3AIllinois/Hybrid_Promoter)
* [Modeling](https://2009.igem.org/Team%3AIllinois/Modelingteam)

## **Post-Summer Progress**

This section includes entries for lab work done after changes were made to the way the project was handled in lab when summer ended.

## **September 22**

PCRed the plasmids containing the Lac promoter, the Tet promoter, CFP, YFP, and GFP. The gel indicated poor results. We found that the primers for the fluorescent protein genes can anneal on their overhang as well. We will need to order new primers for these genes.

## **September 24**

PCRed all target sequences w/ BB sRNAs w/ no results

## **September 25**

PCRed sRNA genes (MicC, MicA, MicF) and their respective target sequences (OmpC, OmpA, OmpF). The gel was run with the Vogel primers as a control since the previous attempt to PCR Biobricks failed.

Also PCRed plasmid pSB1A2. Used 56°C for annealing temperature instead of 58°C and got better results.

Also restreaked plates of E. coli containing plasmid PJU-334 and Biobricks BBa\_K091101, BBa\_I0500, and BBa\_R0040.

## **September 26**

PCRed sRNA target sequences OmpC, OmpA, OmpF, FtsZ, PtsG, GalK,. For PCRs, ran a 50 μL reaction using

* 4 μL chromosomal template
* 0.4 μL of each primer
* 5 μL 10x buffer
* 0.8 μL Pfu polymerase
* 1 μL dNTP mix
* 38.4 μL dH2O

Also digested OmpC, OmpA, and OmpF target sequences from yesterday's PCR. The first round of digestions was run for three hours using

* 1 μL EcoRI-HF (20,000 U/mL)
* 3.5 μL 10x Buffer 4
* 30 μL DNA
* 0.35 μL BSA
* 0.15 μL dH2O

The second round of digestions used

* 0.2 μL PstI (100,000 U/mL)
* 3.5 μL 10x Buffer 3
* 30 μL DNA
* 0.35 μL BSA
* 1 μL dH2O

Also cultured cells containing plasmid PJU-334 and Biobricks BBa\_K091101, BBa\_I0500, and BBa\_R0040 overnight.

## **September 27**

Miniprepped cells containing plasmid PJU-334 and Biobricks BBa\_K091101, BBa\_I0500, and BBa\_R0040. Also digested target sequences from yesterday. For the first round of digestions, ran for three hours using

* 0.4 μL PstI (100,000 U/mL)
* 3.5 μL 10x Buffer 3
* 30 μL DNA
* 0.35 μL BSA
* dH2O to 35 μL

For the first round of digestion, the incorrect buffer may have been used. The digestion was then cleaned up by PCR cleanup, and then the second round of digestions were run for 2.5 hours using

* 2 μL EcoRI-HF (20,000 U/mL)
* 4 μL 10x Buffer 4
* 30 μL DNA
* 0.4 μL BSA
* 3.6 μL dH2O

## **September 28**

PCRed the Lac promoter, the Tet promoter (BBa\_R0040), and the Tet promoter-RBS construct in a 100 μL reaction using

* 2 μL template
* 0.8 μL each primer
* 20 μL 5x Phusion HF Buffer
* 2 μL dNTP mix
* 0.6 μL Phusion polymerase
* 73.8 μL dH2O

Also performed dpnI digestion (3 μL) for 4.75 hours.

## **September 29**

PCRed sRNA genes DicF, gcvB, and SgrS out of the E. coli chromosome in 50 μL reactions using

* 4 μL template
* 0.4 μL each primer
* 5 μL 10x buffer
* 1 μL dNTP mix
* 0.8 μL Pfu polymerase
* 38.4 μL dH2O

## **September 30**

Digested the Lac promoter, Tet promoter, and Tet promoter-RBS construct from the September 28 PCR for four hours using

* 0.4 μL PstI (100,000 U/mL)
* 30 μL DNA
* 3.5 μL BSA
* 0.5 μL dH2O

Also ligated and transformed sRNA genes into plasmid PJU-334, but did not see any colonies.

## **October 1**

Digested sRNA genes DicF, SgrS, and GcvB for 2:20 hours using

* 0.4 μL PstI (100,000 U/mL)
* 3.5 μL 10x Buffer 3
* 30 μL DNA (29 μL for DicF)
* 0.35 μL BSA
* dH2O to 3 μL

Performed a PCR cleanup on reaction afterwards.

Also PCRed GFP, CFP, and YFP genes using

* 1 μL template
* 0.4 μL each primer
* 10 μL 5x Phusion HF Buffer
* 1 μL dNTP mix
* 0.3 μL Phusion polymerase
* 36.9 μL dH2O

## **October 2**

Ligated target sequences of OmpA, OmpC, and OmpF into plasmid pXG-10:

|  |  |  |
| --- | --- | --- |
| **OmpA:** | **OmpC:** | **OmpF:** |
| * 2 μL vector (19 μg/μL) * 0.5 μL insert (40 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 14.5 μL dH2O | * 1 μL vector (19 μg/μL) * 1.3 μL insert (40 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 14.7 μL dH2O | * 2 μL vector (19 μg/μL) * 1 μL insert (40 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 14 μL dH2O |

Ligated for 1:40 hours, transformed by heat shock by putting cells on ice for 30 minutes, in 42°C bath for 45 seconds, and 1 hour recovery.

## **October 3**

The ligations were unsuccessful. No colonies appeared on the plates.

## **October 4**

Performed colony boil of MG1655 for 10 minutes at 99deg;C in thermocycler in 150 μL water with a 5 minute spin down. The concentrations received were low (8 ng/μL).

PCRed sRNA genes and plasmid PJU-334:

|  |  |
| --- | --- |
| **sRNA Genes:** | **PJU-334:** |
| * 6 μL chromosomal template (8 ng/μL) * 0.4 μL each primer * 5 μL Pfu buffer * 1 μL dNTP mix * 0.8 μL Pfu polymerase (used turbo on all but MicF) * 36.4 μL dH2O | * 1 μL template (112 ng/μL) * 0.8 μL each primer * 20 μL 5x Phusion HF Buffer * 2 μL dNTP mix * 0.6 μL Phusion polymerase * 74.8 μL dH2O |

After PCR, digested PJU-334 with dpnI for 4-5 hours, PCR-purified reaction afterwards.

Ligated 6 sRNA genes into Biobrick plasmids and 3 target sequences into plasmid pXG-10 using an insert-to-vector ratio of 5:1 for blunt-end sRNAs and a ratio of 3:1 for normal sticky-end sRNAs. Used 48:20 ratio for target sequences at 5'-UTR (as in Vogel paper)

|  |  |  |  |
| --- | --- | --- | --- |
| **MicC:** | **DicF:** | **GcvB:** | **SgrS:** |
| * 2 μL vector (23 μg/μL) * 2 μL insert (9 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 13 μL dH2O | * 2 μL vector * 0.85 μL insert (14.5 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 14.15 μL dH2O | * 2 μL vector * 1.75 μL insert (14 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 13.25 μL dH2O | * 2 μL vector * 2.3 μL insert (13 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 12.7 μL dH2O |
| **MicA:** | **MicF:** | **OmpA:** | **OmpC:** | **OmpF:** |
| * 2 μL vector * 1.3 μL insert (11 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 13.7μL dH2O | * 2 μL vector * 1.4 μL insert (14 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 13.6 μL dH2O | * 3.7 μL vector * 0.55 μL insert * 2 μL 10x ligase buffer * 1 μL ligase * 16.45 μL dH2O | * 3.7 μL vector * 2.2 μL insert * 2 μL 10x ligase buffer * 1 μL ligase * 14.8 μL dH2O | * 3.7 μL vector * 0.7 μL insert * 2 μL 10x ligase buffer * 1 μL ligase * 16.3 μL dH2O |

Ligated for 2 hours at room temperature, transformed by heat shock (cells 30 minutes on ice, 45 seconds in 42°C bath, 4 minutes on ice, recover in 450 μL LB in culture tubes at 37°C for 1 hour), centrifuged culture for 5 minutes and resuspended in 100 μL water, plated all of it.

## **October 5**

The sRNA gene ligations were successful, but the sRNA target sequence ligations were not.

Digested sRNA genes and plasmid PJU-334 from PCR on October 4, as well as plasmid pXG-10 (pXG-10 was digested w/ one enzyme for 5 hours, PCR-purified, and digested with the second enzyme for 13.5 hours overnight):

|  |  |  |  |
| --- | --- | --- | --- |
| **sRNA Genes:** | **PJU-334:** | **pXG-10 (1st round):** | **pXG-10 (2nd round):** |
| * 30 μL DNA * 0.2 μL XbaI (100,000 U/mL) * 0.35 μL BSA * 3.5 μL Buffer 4 * 1 μL dH2O | * 30 μL DNA * 0.3 μL XbaI (100,000 U/mL) * 0.35 μL BSA * 3.5 μL Buffer 4 * 0.9 μL dH2O | * 21 μL DNA * 1.5 μL NheI (20,000 U/mL) * 0.3 μL BSA * 3.0 μL Buffer 4 * 4.2 μL dH2O | * 30 μL DNA * 3 μL NsiI * 0.4 μL BSA * 4.0 μL Buffer 3 * 2.6 μL dH2O |

An analysis gel on the PJU-334 digestion showed that it worked very well!

Set up overnight cultures of cells transformed with our Biobricks:

* MicA (1,2,3)
* MicF (1,2,3)
* MicC (1,2,3)
* GcvB (1,2,3)
* SgrS (1,2,3)
* DicF (1,2,3)
* pXG-10 (3)
* GFP for submission (2)

## **October 6**

All overnight cultures were miniprepped. Glycerol stocks were also made.

Digested GFP and pXG-10 minipreps. The first round for both went for 3:50 hours, and the second round for both went for 4:20 hours:

|  |  |  |  |
| --- | --- | --- | --- |
| **GFP-Vector (1st round):** | **GFP-Vector (2nd round):** | **pXG-10 (1st round):** | **pXG-10 (2nd round):** |
| * 30 μL DNA * 0.4 μL PstI * 0.4 μL BSA * 4 μL Buffer 3 * 5.2 μL dH2O | * 29 μL DNA * 2 μL EcoRI-HF * 0.4 μL BSA * 4 μL Buffer 4 * 4.6 μL dH2O | * 30 μL DNA * 3 μL NsiI * 0.4 μL BSA * 4.0 μL Buffer 3 * 2.6 μL dH2O | * 30 μL DNA * 2 μL NheI-HF * 0.4 μL BSA * 4.0 μL Buffer 4 * 3.6 μL dH2O |

Ligated sRNA genes into plasmid PJU-334:

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **MicC:** | **MicF:** | **MicA:** | **SgrS:** | **GcvB:** |
| * 1.5 μL vector * 2.6 μL insert (11 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 12.9μL dH2O | * 1.5 μL vector * 1.3 μL insert (14 μg/μL) * 2 μL 10x ligase buffer * 1 μL ligase * 14.2 μL dH2O | * 1.5 μL vector * 1.1 μL insert * 2 μL 10x ligase buffer * 1 μL ligase * 14.4 μL dH2O | * 1.5 μL vector * 4.8 μL insert * 2 μL 10x ligase buffer * 1 μL ligase * 10.7 μL dH2O | * 1.5 μL vector * 4.9 μL insert * 2 μL 10x ligase buffer * 1 μL ligase * 10.6 μL dH2O |

Ligated for 2:20 hours at room temperature. Transformed by heat shock (33 minutes on ice, 45 seconds in 42°C bath, 3 minutes on ice, recovery for 1 hour in 450 μL LB in culture tube), plated 100 μL/plate.

## **October 7**

Overnight cultures of MicA, MicC, MicF, SgrS, and GcvB. Also plated rest of recovery from yesterday.

## **October 17**

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