iGEM 2011 — Team Hong_Kong-CUHK

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# Team:Hong Kong-CUHK/Notebook

### From 2011.igem.org

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Chinese University of Hong Kong iGEM 2011

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---

![](http://www.cse.cuhk.edu.hk/~zwang9/igem/img/lab.png)

* [Note Book](/Team%3AHong_Kong-CUHK/Notebook)
* [Protocol](/Team%3AHong_Kong-CUHK/Laboratory/protocol)
* [Collaboration](/Team%3AHong_Kong-CUHK/Laboratory/collaboration)
* [Biobricks construction](/Team%3AHong_Kong-CUHK/Laboratory/biobricks)

### 2011 CUHK iGEM Note Book

➨ Select week number from the table above

**Week 0**

**2 June2011 (Tue)**

**1.** **Frankie- transformed the bacteria with RFP and prepared the T7 and HR gene plasmid for PCR.**

**Week 1** **Monday 6 June – Sunday 12 June**

**8 June2011 (Wed)**

**1.** **Test the effect of different concentration of NaCl to the survival of bacterial** **DH5****a****& BL21**

**9 June2011 (Thu)**

**1.** **Repeat Wed work**

**2.** **IPTG inducer**

**3.** **PCR amplification - T7 and HR(rhodopsin)**

**4.** **Gel clean to get T7 and HR gene from gel**

**10 June2011 (Fri)**

**1.** **Restriction cut of T7, HR and iGEM vector, ligate them together**

**2.** **Received all medium to grow magnetobacteria**

**Week 2** **Monday 13 June – Sunday 19 June**

**13 June 2011 (Mon)**

**1.** **Nanodrop test DNA concentration**

**2.** **Transformation to competent cells, spread on plate A & plate K**

**14 June 2011 (Tue)**

**1.** **no colonies were found on spread plate. possible reasons:**

Ø   antibiotic C resistance spread on plate K

Ø   failed restriction cut,

Ø   spreading tools too hot which kill the bacteria…

**2.** **A****ttempt to mix culturing medium for halobacterium salinarum DSM 3754, haloterrigena turkmenica DSM 5511, Natronomonas pharaohs DSM 2160, but couldn't find casamino acids which are essential for all the medium required**

**3.** **Repeat work of last week: restriction cut, ligation**

**15 June 2011 (Wed)**

**1.** **transformation o****f** **E.coli competent cells.**

****Week 3** **Monday 20 June – Sunday 26 June**

**20 June (Mon)**

**1.** **Restriction cut**

Ø   pSB1A3 & pSB1K3

Ø   HR & T7 promoter

Ø   restriction enzyme: EcoR1 &Pst1

Ø   incubate at 37oC , 2hours

**2.** **PCR purification of cut product**

Ø   refer to kit protocol

**3.** **Agarose gel electrophoresis (Run gel) of purified product**

Ø   at 120V, 45minutes

Ø   Failed. Possible reasons: insufficient DNA conc. In gel, stock problem

**4.** **Ligation**

Ø   HR+pSB1A3

Ø   T7+ pSB1A3

Ø   HR+ pSB1K3

Ø   T7+ pSB1K3

Ø   at 16 oC, overnight

**5.** **Inoculation transformation buffer preparation**

Ø   Steps of preparing 500ml inoculation transformation buffer

1.      55mM MaCl2‧4H2O (5.4425g)

2.      15mM CaCl2‧2H2O (1.1025g)

3.      250mM KCl (9.3189g)

4.      10mM PIPES (10ml) (taken from 4oC fridge)

5.      Stored in 4oC fridge

Ø   109 plate with antibiotic A and K is poured

**6.** **Detection of T7 and HR by gel electrophoresis**

Ø   no band of sample is shown

**21 June (Tue) No record**

**22 June (Wed)**

**1.** **Pick colonies**

Ø   Each plate pick 2 colonies into 2 tubes

**2.** **Inoculation**

Ø   at 37 oC, 250rpm, 7hours

**3.** **Transfer**

Ø   DH5α(B tube) : 1ml & 0.5 ml

Ø   BL 21 (B tube) : 1ml & 0.5 ml

Ø   Rosetta (A tube): 2ml

Ø   Overnight, 200rpm

**4.** **Prepare 500ml LB, 500ml LB with 1M NaCl and 500ml LB with 1M KCl solution LB solution**

**5.** **transform pET27bHR with K resistance into DH5α, BL21**

**23 June (Thu)**

**1.** **Measure OD of transfer**

Ø   All tubes’ OD > 0.55

Ø   Due to contamination in the step of picking colonies. We shouldn’t put the pipette tips into the tubes since our medium does not contain antibodies

**2.** **Repeated the experiment from picking colonies and inoculation**

Ø   Only DH5α is used

Ø   7 hours inoculation

Ø   Inoculation failed.

**3.** **Restarted from culturing E.coli on agar plate**

Ø   DH5α & BL21

**4.** **Restriction cut and Double Digestion**

Ø   HR   20ng/ul

Ø   T7   20ng/ul

Ø   Since there are not enough solution(a 50ul system should have at least 200ng of DNA) to make up a 50ul system, we modify the system to 25ul.

|  |  |
| --- | --- |
| Solution (added by order) | Amount(ul) |
| ddH20 | 14 |
| 10X Buffer 3 | 2.5 |
| 10X BSA | 2.5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| HR | 5 |
| **Total** | **25ul** |

|  |  |
| --- | --- |
| Solution (added by order) | Amount(ul) |
| ddH20 | 14 |
| 10X Buffer 3 | 2.5 |
| 10X BSA | 2.5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| T7 | 5 |
| **Total** | **25ul** |

|  |  |
| --- | --- |
| Solution (added by order) | Amount(ul) |
| ddH20 | 15 |
| 10X Buffer 3 | 2.5 |
| 10X BSA | 2.5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| pSB1 | 4 |
| **Total** | **25ul** |

Ø   The 3tubes are put at 37 oC for 2hours

**5.** **Restriction cut**

Ø   T7/HR restriction cut mixture

Ø   pSB1 T3 restriction cut mixture

Ø   The mixture is put into thermomixer at 37oc for 2hs

Ø   Inoculated 5ml start culture of DH5α,, no BL21 colony is observed on the plate

Ø   6 sets of solution is prepared

0 – 1M NaCl with IPTG

0 – 1M NaCl without IPTG

0 – 1M KCl with IPTG

0 – 1M KCl without IPTG

0M salt with IPTG

0M salt without IPTG

(the solution without salt is set as controls)

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| [Salt]/M | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.5 | 0.6 | 0.7 | 0.8 | 0.9 | 1.0 |
| LB + salt/ml | 0 | 0.4 | 0.8 | 1.2 | 1.6 | 2.0 | 2.4 | 2.8 | 3.2 | 3.6 | 4 |
| LB/ml | 4.0 | 3.6 | 3.2 | 2.8 | 2.4 | 2.0 | 1.6 | 1.2 | 0.8 | 0.4 | 0 |

Ø   For each tube, 2ul IPTG is added (for sample with IPTG), 40ul DH5α start culture, 4ml LB+ salt volumes

Ø   5ml start culture of DH5αis inoculated again for the miniprep on next day

Ø   OD of solution is checked on next day

**24 June (Fri)**

**1.** **Picking E.coli colonies cultured yesterday**

Ø   DH5α & BL21

**2.** **Inoculation:**

Ø   4 DH5α tubes & 4 BL21 tubes

Ø   Each with 5ml LB

Ø   37℃, 250rpm

**3.** **Transfer inoculation mixture into 125ml LB in conical flask**

Ø   1.0ml DH5α-2, 0.5ml DH5α-2

Ø   1.0ml BL21-2, 0.5ml BL21-2

Ø   Shake at 37℃, 250rpm, overnight

**4.** **Transform “HR+1T3” & “T7+1T3” into DH5α competent cells**

Ø   1.0ml HR+1T3, 0.5ml HR+1T3

Ø   1.0ml T7+1T3, 0.5ml T7+1T3

Ø   1 control plate without antibiotics tetracycline

Ø   Incubate at 37℃, overnight

**5.** **Light absorbance of DH5α KCl IPTG**

Ø   DH5α KCl, DH5α NaCl IPTG and DH5α NaCl is measured

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α KCl IPTG | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(5x) | 0.532 | 2.660 |
| 0.2(5x) | 0.515 | 2.575 |
| 0.3(5x) | 0.477 | 2.385 |
| 0.4(5x) | 0.390 | 1.950 |
| 0.5(5x) | 0.370 | 1.850 |
| 0.6(5x) | 0.267 | 1.335 |
| 0.7(5x) | 0.92 | 0.960 |
| 0.8(2x) | 0.300 | 0.600 |
| 0.9(2x) | 0.156 | 0.312 |
| 1.0 | 0.249 | 0.249 |
| 0 | 0.791 | 0.791 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α KCl | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(5x) | 0.500 | 2.500 |
| 0.2(5x) | 0.447 | 2.235 |
| 0.3(5x) | 0.430 | 2.150 |
| 0.4(5x) | 0.399 | 1.995 |
| 0.5(5x) | 0.305 | 1.525 |
| 0.6(5x) | 0.264 | 1.32 |
| 0.7(5x) | 0.205 | 1.025 |
| 0.8(5x) | 0.117 | 0.585 |
| 0.9(2x) | 0.220 | 0.440 |
| 1.0 | 0.263 | 0.263 |
| 0(5x) | 0.519 | 2.595 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α NaCl IPTG | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(2x) | 0.829 | 1.658 |
| 0.2(2x) | 0.844 | 1.688 |
| 0.3(5x) | 0.898 | 1.796 |
| 0.4(5x) | 0.384 | 1.920 |
| 0.5(5x) | 0.340 | 1.700 |
| 0.6(5x) | 0.208 | 1.040 |
| 0.7(2x) | 0.393 | 0.786 |
| 0.8(5x) | 0.287 | 0.574 |
| 0.9(2x) | 0.119 | 0.238 |
| 1.0 | 0.113 | 0.113 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α NaCl | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(5x) | 0.499 | 2.495 |
| 0.2(2x) | 0.478 | 2.390 |
| 0.3(5x) | 0.513 | 2.565 |
| 0.4(5x) | 0.390 | 1.950 |
| 0.5(5x) | 0.302 | 1.510 |
| 0.6(5x) | 0.246 | 1.230 |
| 0.7(5x) | 0.194 | 0.970 |
| 0.8(2x) | 0.269 | 0.538 |
| 0.9 | 0.284 | 0.284 |
| 1.0 | 0.103 | 0.103 |

**6.** **Miniprep**

**7.** **Storage of cells**

Ø Transfer 1ml from each cell culture to 1.5ml microcentrifuge tube

Ø Spin for 1min

Ø Remove medium by discarding and pipetting

Ø Store the cells at -80oc

**25 June (Sat)**

**1.** **OD measurement (OD 600nm) of transferred product**

|  |  |  |
| --- | --- | --- |
| BL21 | 0.5ml | 1.891 |
| BL21 | 1.0ml | 1.831 |
| DH5α | 0.5ml | 0.670 |
| DH5α | 1.0ml | 1.415 |

Ø   All tubes’ OD > 0.55

Ø   Might have contamination

Ø   Still used DH5α 0.5ml 0.670 OD600 to make competent cells

Ø   Made 25 tubes of competent cells at -80℃

**2.** **pick colonies, Inoculation,**

Ø   2 from HR+1T3, 2 from T7+1T3 II, 1 from HR+1T3 I into 5ml LB+tetracycline snap-cap tube

**3.** **Transfer 125ml magnetotactic bacteria medium to a conical flask, waiting for autoclave****

**Week 4** **Monday 27 June – Sunday 3 July**

**27 June (Mon)**

**1.** **Inoculated 5ml start culture of DH5α transformed with HR pET37b using the plate the week before**

**2.** **Prepared 6 conical flasks**

Ø LB with 50ul IPTG + 100ul antibiotic K

Ø LB without IPTG + 100ul antibiotic K

Ø 0.6M NaCl LB with 50ul IPTG + 100ul antibiotic K

Ø 0.6M NaCl LB without IPTG + 100ul antibiotic K

Ø 0.6M KCl LB with 50ul IPTG + 100ul antibiotic K

Ø 0.6M KCl LB without IPTG + 100ul antibiotic K

Ø For measuring OD absorbance and plot growth curve of DH5α on 28/06/2011 every 30/45min

**3.** **Prepared 4 sets of solution same as 23/06/2011 to verify the optimum concentration of salt for** **DH5α to grow and absorb salt and added DH5α start culture**

**4.** **Inoculate 5ml start culture of** **DH5α transformed with HR pET27b using the plate of the week before for the 6 conical flasks for 8/06/2011 growth curve (+5ul antibiotic K)**

**28 June (Tue)**

**1.** **Prepare another set of competent cell**

Ø   Pick single colony from plates prepared on Sat

Ø   Transfer the colony into 5ml of LB broth in FALCON

Ø   Incubate for 6hrs @37℃, shaking (250-300rpm)

Ø   ~6pm, use the incubated starter culture to prepare 2 1L flasks

|  |  |
| --- | --- |
| FLASK1: 0.5ml culture + 125LB | FLASK2: 0.25 ml culture + 125LB |

Ø   Incubate at 22℃, moderate shaking, overnight

**2.** **Transform competent cells prepared on Sat 25 June**

Ø   Incubate at 37℃, overnight

**3.** **Plasmid DNA purification**

Ø   T7, PSB1K3 / HR, PSB1K3

Ø   USING Spin MiniPrep Kit and a Microcentrifuge

**4.** **Light absorbance of** **DH5α KCl IPTG, DH5α KCl, DH5α NaCl IPTG, DH5α NaCl, DH5α 0M LB and DH5α 0M LB IPTG**

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α KCl IPTG | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(2x) | 0.916 | 1.832 |
| 0.2(2x) | 0.756 | 1.512 |
| 0.3(2x) | 0.527 | 1.054 |
| 0.4 | 0.426 | 0.426 |
| 0.5 | 0.710 | 0.710 |
| 0.6 | 0.310 | 0.310 |
| 0.7 | 0.156 | 0.156 |
| 0.8 | 0.129 | 0.129 |
| 0.9 | 0.072 | 0.072 |
| 1.0 | 0.012 | 0.012 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α KCl | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1 | 0.618 | 2.472 |
| 0.2 | 0.427 | 1.708 |
| 0.3 | 0.274 | 1.096 |
| 0.4 | 0.845 | 1.690 |
| 0.5 | 0.474 | 0.948 |
| 0.6 | 0.970 | 0.970 |
| 0.7 | 0.626 | 1.252 |
| 0.8 | 0.504 | 0.504 |
| 0.9 | 0.083 | 0.083 |
| 1.0 | -0.011 | -0.011 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α NaCl IPTG | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(2x) | 0.794 | 1.588 |
| 0.2(2x) | 0.716 | 1.432 |
| 0.3(2x) | 0.483 | 0.966 |
| 0.4(2x) | 0.436 | 0.872 |
| 0.5 | 0.761 | 0.761 |
| 0.6 | 0.713 | 0.713 |
| 0.7 | 0.641 | 0.641 |
| 0.8 | 0.491 | 0.491 |
| 0.9 | 0.168 | 0.168 |
| 1.0 | 0.044 | 0.044 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α NaCl | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1(2x) | 0.932 | 1.864 |
| 0.2(2x) | 0.719 | 1.438 |
| 0.3(2x) | 0.535 | 1.070 |
| 0.4(2x) | 0.525 | 1.050 |
| 0.5 | 0.882 | 0.882 |
| 0.6 | 0.819 | 0.819 |
| 0.7 | 0.712 | 0.712 |
| 0.8 | 0.470 | 0.470 |
| 0.9 | 0.123 | 0.123 |
| 1.0 | 0.031 | 0.031 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α 0M LB | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1 | 0.763 | 1.526 |
| 0.2 | 0.829 | 1.658 |

Ø   0.8ml DH5α is added to start culture solution of the 6 conical flask prepared the day before, the absorbance of solution is measured per 30mins

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Time(h)\OD of Solution | LB | LB IPTG | 0.6M NaCl LB | 0.6M NaCl LB IPTG | 0.6M KCl LB | 0.6M KCl LB IPTG |
| 0 | 0.015 | 0.016 | 0.028 | 0.030 | 0.025 | 0.047 |
| 0.5 | 0.025 | 0.024 | 0.050 | 0.017 | 0.041 | 0.014 |
| 1 | 0.035 | 0.039 | 0.077 | 0.015 | 0.025 | 0.029 |
| 1.5 | 0.071 | 0.080 | 0.070 | 0.005 | 0.036 | 0.026 |
| 2 | 0.146 | 0.176 | 0.111 | 0.005 | 0.044 | 0.044 |
| 2.5 | 0.307 | 0.338 | 0.148 | 0.003 | 0.063 | 0.057 |
| 3 | 0.473 | 0.527 | 0.262 | 0.017 | 0.093 | 0.093 |
| 3.5 | 0.650 | 0.669 | 0.345 | 0.012 | 0.134 | 0.137 |
| 4 | 0.874 | 0.888 | 0.473 | 0.003 | 0.219 | 0.210 |
| 4.5 | 10.66 | 1.128 | 0.626 | 0.001 | 0.323 | 0.310 |
| 5 | 0.753(2x) | 0.800(2x) | 0.722 | 0.001 | 0.386 | 0.365 |

Ø   5ml start culture of DH5α is transformed with HR pET27b (using the plate the week before) is inoculated, 6 conical flasks of solution is prepared same as the 12/06/2011

**29 June (Wed)**

**1.** **Measure OD of competent cell prepared yesterday**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Flask** | **1** | **2** | **3** | **4** |
| **OD 600** | **1.697** | **1.844** | **1.840** | **1.662** |

Ø   All flasks have OD greater than 0.55, it indicates contamination.

Ø   Failed

**2.** **Measure DNA concentration**

Ø   Use DNA purified yesterday

|  |  |  |
| --- | --- | --- |
|  | 260/280 | DNA concentration (ng/ul) |
| T7 pSB1K3 (1) | 1.95 | 32.6 |
| T7 pSB1K3 (2) | 1.90 | 32.2 |
| HR pSB1K3 (1) | 2.08 | 12.2 |
| HR pSB1K3 (2) | 2.01 | 12.3 |

**3.** **Prepare new competent cells**

Ø   Transfer 0.1 nd 0.5ml DH5a and BL21 into 125ml LB

**4.** **Double restriction cut of T7 pSB1K3 and HR pSB1K3**

|  |  |
| --- | --- |
| Water | 11.75ul |
| Buffer 3 | 3.5ul |
| BSA | 0.35ul |
| EcoR1 | 0.7ul |
| Pst1 | 0.7ul |
| T7 pSB1K3 | 8ul |
| Total | 25ul |

|  |  |
| --- | --- |
| Buffer 3 | 2.5ul |
| BSA | 0.25ul |
| EcoR1 | 0.5ul |
| Pst1 | 0.5ul |
| HR pSB1K3 | 21.25ul |
| Total | 25ul |

Ø   Incubate at 37oC for 2hrs

**5.** **Run gel**

Ø   T7: band at ~150kb

Ø   HR: band at ~800kb

**6.** **The absorbance of 6 solutions**

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Time(h)\OD of Solution | LB | LB IPTG | 0.6M NaCl LB | 0.6M NaCl LB IPTG | 0.6M KCl LB | 0.6M KCl LB IPTG |
| 0 | 0.002 | 0.023 | 0.019 | 0.026 | 0.001 | 0.001 |
| 0.5 | 0.006 | 0.004 | 0.020 | 0.032 | 0.003 | 0.018 |
| 1 | 0.012 | 0.022 | 0.043 | 0.031 | 0.011 | 0.006 |
| 1.5 | 0.023 | 0.028 | 0.047 | 0.021 | 0.017 | 0.032 |
| 2 | 0.110 | 0.151 | 0.065 | 0.068 | 0.025 | 0.021 |
| 2.5 | 0.279 | 0.345 | 0.129 | 0.075 | 0.029 | 0.053 |
| 3 | 0.430 | 0.455 | 0.488 | 0.137 | 0.068 | 0.057 |
| 3.5 | 0.591. | 0.640 | 0.230 | 0.183 | 0.089 | 0.112 |
| 4 | 0.748 | 0.830 | 0.310 | 0.264 | 0.122 | 0.150 |
| 4.5 | 0.962 | 1.053 | 0.445 | 0.353 | 0.183 | 0.265 |
| 5 | 0.747 | 0.832 | 0.389 | 0.310 | 0.177 | 0.209 |
| 5.5 | 0.939 | 10.17 | 0.448 | 0.380 | 0.235 | 0.261 |
| 6 | 0.764 | 0.745 | 0.360 | 0.316 | 0.251 | 0.310 |

**30 June (Thur)**

**1.** **Measure OD of 0.1 and 0.05ml of DH5a and BL21 in 125 LB prepared on 29 June**

|  |  |  |
| --- | --- | --- |
|  | Volume (ul) | OD 600 |
| DH5a | 50 | 0.352 |
|  | 100 | 0.523 |
| BL 21 | 50 | 0.349 |
|  | 100 | 1.710 |

Ø   DH5a 100ul is taken for making competent cells

Ø   Use competent cells made to transform T7 and HR

Ø   Streak plates to check if competent cells viable

Ø   Total of 8 plates

Ø   Incubate at 37oC overnight

**2.** **Spread plates with transformed competent cells**

Ø   T7 pSB1K3 in DH5a

Ø   HR pSB1K3 in DH5a

Ø   Total of 4 plates

**3.** **PCR purification of iGEM plasmid**

**4.** **PCR purification of plasmid made**

Ø   1K3, 1T3, 1C3

**5.** **Run gel**

Ø   No result

**1 July (Fri)**

**1.** **Check plates**

Ø   All 8 plates shown bacteria growth

Ø   All 4 plates shown transformed cells growth

**2.** **Pick colonies**

Ø   DH5a and competent cells with T71K3 and HR1K3

Ø   Total of 12 snap caps

Ø   Shake at 250rpm, 37oC overnight

**3.** **PCR amplification and purification of iGEM psB1K3**

**4.** **Run gel**

Ø   No banding

**2 July (Sat)**

**1.** **Mini prep**

Ø   DH5a HR                               x3

Ø   DH5a T7                                x3

Ø   HR1K3                                   x3

Ø   T71K3                                    x2

Ø   One of the T71K3 didn’t form pallet after centrifugation

**2.** **Digestion**

|  |  |
| --- | --- |
| Water | 11.2ul |
| DNA | 10ul |
| Buffer 3 | 2.5ul |
| EcoR1 | 0.5ul |
| Pst1 | 0.5ul |
| BSA | 0.25ul |
| Total | 25ul |

Ø   Incubate at 37oC, 2hrs

Ø   Remaining samples are stored at -20oC

**3.** **Run gel (with 2 setups)**

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Well | 1 | 2 | 3 | 4 | 5 | 6 |
|  | ladder | T71K3 (1) | T71K3 (2) | HR(1) | HR(2) | HR(3) |

Ø   Result:           Lane 1 and 3 have bands of ~100-200bp

Land 4 to 6 has bands of 800bp

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Well | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
|  | ladder | T7(1) | T7(2) | T7(3) | HR1K3(1) | HR1K3(2) | HR1K3(3) |

Ø   Result:           Lane 2 to 4 has bands of ~100-200bp

Lane 5 to 7 has bands of ~800bp

**3 July (Sun): No record**

**Week 5** **Monday 4 July – Sunday 10 July**

**4 July (Mon)**

**1.** **Pick colonies**

Ø   BL21, 3 snap caps

Ø   Incubate at 37oC, 250rpm

**1.** **Prepare plates with antibiotics**

Ø   25 plates with C

Ø   43 plates with K

**2.** **Transfer incubated BL21 to 125ml LB, total of 3 samples, each with 50ul BL21 added**

Ø   Incubate at37oC overnight, 150rpm

**3.** **5ml start culture of DH5α is inoculated and put into the incubator in common room**

**4.** **6 conical flasks is prepared**

Ø   LB IPTG

Ø   0.6M NaCl LB IPTG

Ø   0.6M KCl LB IPTG

Ø   0.1M – 1M NaCl IPTG

Ø   0.1M – 1M KCl IPTG

Ø   LB without IPTG

**5 July (Tue)**

**1.** **Prepare competent cells**

|  |  |
| --- | --- |
|  | OD |
| BL21 (1) | 1.958 |
| BL21 (2) | 1.918 |
| BL21 (3) | 1.907 |

Ø   OD greater than 0.55, repeat the experiment

Ø   Using BL21, we incubate cells from picking colony from plates

Ø   Culture from tubes are transferred to the flask containing LB at 25oC overnight, 130 rpm

**2.** **PCR purification of T71K3 plasmid**

Ø   Using iGEM protocol

Ø   Concentration of primers is 100pmol/ul, hence we have to dilute it to 30pmol/ul

**3.** **Run gel using iGEM protocol and product protocol**

Ø   After PCR amplification

Result: both show bandings beyond 100bp

iGEM protocol shows banding at 2000kb

Ø   After PCR purification

Result: iGEM protocol shows banding at 2000kb

**4.** **Inoculation**

**5.** **1ml DH5α start culture solution is added to3 conical flasks prepared on 4/7.**

Ø   The absorbance and cell density is measured every 30 minutes.

Ø   Cell density at 2h and 2.5 h is failed to be measured.

Ø   Procedure of measuring cell density

i.                1ml solution is transferred to microcentrifuge tube, centrifuge for 1min and resuspend

ii.                10ul solution is transfer to both side of the hematocytometer and cover with the cover slit

iii.                observe the central square with 10x magnification under microscope and count the number of cell

Ø   Dilution is required (10fold, 100fold, 1000fold .. ) for higher absorbance

Ø   (1OD unit = 109 cell)

Ø   Absorbance of 21 tubes is measured.

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α NaCl IPTG( 4times diluted) | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1 | 0.701 | 2.804 |
| 0.2 | 0.786 | 3.144 |
| 0.3 | 0.680 | 2.720 |
| 0.4(2x) | 0.947 | 3.788 |
| 0.5(2x) | 0.603 | 2.412 |
| 0.6(2x) | 0.538 | 2.152 |
| 0.7 | 0.334 | 1.336 |
| 0.8 | 0.083 | 0.332 |
| 0.9 | 0.025 | 0.100 |
| 1.0 | 0.022 | 0.088 |

|  |  |  |
| --- | --- | --- |
| Light absorbance of DH5α KCl IPTG( 4times diluted) | | |
| Concentration/M | OD (ABS/cm) | Final OD (ABS/cm) (without dilution) |
| 0.1 | 0.893 | 3.572 |
| 0.2 | 0.906 | 3.624 |
| 0.3 | 0.780 | 3.120 |
| 0.4 | 0.445 | 1.780 |
| 0.5 | 0.621 | 2.484 |
| 0.6 | 0.083 | 0.332 |
| 0.7 | 0.218 | 0.872 |
| 0.8 | 0.040 | 0.160 |
| 0.9 | 0.010 | 0.040 |
| 1.0 | 0.027 | 0.108 |

Ø   Light absorbance of 3 solutions

|  |  |  |  |
| --- | --- | --- | --- |
| Time(h)\OD of Solution | LB IPTG | 0.6M NaCl LB | 0.6M KCl LB |
| 0 | 0.025 | 0.070 | 0.054 |
| 0.5 | 0.043 | 0.065 | 0.044 |
| 1 | 0.117 | 0.068 | 0.067 |
| 1.5 | 0.185 | 0.041 | 0.059 |
| 2 | 0.389 | 0.069 | 0.064 |
| 2.5 | 0.634 | 0.085 | 0.081 |
| 3 | 1.039 | 0.107 | 0.111 |
| 3.5 | 1.586 | 0.188 | 0.181 |
| 4 | 0.334 | 0.274 | 0.268 |
| 4.5 | 0.364 | 0.350 | 0.328 |
| 5 | 2.412 | 0.432 | 0.450 |

Ø   Cell density (HR, IPTG, LBK, DH5α, pET27b HR)

|  |  |
| --- | --- |
| Time(h) | Averaged cell density |
| 0 | 1.5 x 104 |
| 0.5 | 2.0 x 104 |
| 1 | 1.3 x 105 |
| 1.5 | 2.0 x 106 |
| 2 | - |
| 2.5 | - |
| 3 | 8.95 x 106 |
| 3.5 | 8.30 x 109 |
| 4 | 1.145 x 1010 |
| 4.5 | 6.70 x 109 |
| 5 | 6.10 x 109 |

**6 July (Wed)**

**1.** **Prepare BL21 competent cell**

|  |  |
| --- | --- |
| **BL21** | **OD** |
| 1 | 1.094 |
| 2 | 1.263 |
| 3 | 1.263 |
| 4 | 1.163 |

Ø   OD>0.55, preparation failed

**2.** **Restriction cut of T71K3 prepared on 5July**

|  |  |
| --- | --- |
| Buffer 3 | 5ul |
| BSA | 0.5ul |
| Eco R1 | 1ul |
| Pst 1 | 1ul |
| T71K3 | 10ul |

**3.** **Run gel to check the size of 2 sets of T71K3**

Ø   Both samples show banding of ~4000kb

**4.** **Streak plate: BL21 X7**

**5.** **5ml start culture of DH5α is inoculated and put into the incubator in common room**

**6.** **3 conical flasks is prepared**

Ø   LB IPTG

Ø   0.4M NaCl LB IPTG

Ø   0.4M Kcl LB IPTG

**7 July (Thu)****1. Pick colony of BL21 X4 and inoculation**

Ø   37 oC, 250rpm

**2.** **PCR amplification**

Ø   “Home-made” HR1K3, “Home-made” T71K3, stock 1K3

Ø   Using iGEM protocol

|  |  |
| --- | --- |
| PCR supermix | 45ul |
| Upper primer | 1ul |
| Lower primer | 1ul |
| Water | 25ul |
| DNA | 0.5ul |
| TOTAL | 50ul |

**3.** **Transfer 50ul BL21 to 125ul LB**

Ø   Inoculated at 37 oC, 250rpm

**4.** **Run gel**

Ø   Lane1: ladder

Ø   Lane3: “Home-made” HR1K3

Ø   Lane4: “Home-made” T71K3

Ø   Lane5: stock 1K3

**5.** **OD of BL21 in 125ml LB**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 |
| 16:30 | 0.006 | 0.007 | 0.007 | 0.020 |
| 16:45 | 0.007 | 0.013 | 0.01 | 0.007 |
| 20:45 | 0.143 | 0.085 | 0.085 | 0.136 |
| 22:15 | 0.367 |  | 0.312 |  |

**6.** **5ml DH5α start culture solution is added to3 conical flasks prepared on 5/7**

Ø   The absorbance and cell density is measured every 30 minutes

**7.** **3 conical flasks is prepared**

Ø 100ml LB + 50ul IPTG + 100ul antibiotic K

Ø 60ml LB + 40ml KCl LB + 50ul IPTG + 100ul antibiotic K

Ø 60ml LB + 40ml NaCl LB + 50ul IPTG + 100ul antibiotic K

|  |  |  |  |
| --- | --- | --- | --- |
| Time(h)\OD600 of Solution | LB IPTG | 0.4M NaCl LB | 0.4M KCl LB |
| 0 | 0.033 | 0.031 | 0.025 |
| 0.5 | 0.037 | 0.032 | 0.017 |
| 1 | 0.078 | 0.013 | 0.033 |
| 1.5 | 0.122 | 0.056 | 0.047 |
| 2 | 0.310 | 0.102 | 0.096 |
| 2.5 | 0.423 | 0.157 | 0.156 |
| 3 | 0.620 | 0.255 | 0.256 |
| 3.5 | 0.893 | 0.398 | 0.427 |
| 4 | 1.186 | 0.590 | 0.621 |
| 4.5 | 1.632 | 0.859 | 0.803 |

Ø   Cell density of DH5α, pET27b HR, LB IPTG with K

|  |  |
| --- | --- |
| Time(h) | Averaged cell density |
| 0 | 3x106 |
| 0.5 | 2.5 x106 |
| 1 | 3.75 x107 |
| 1.5 | 1 x107 |
| 2 | 9.75 x108 |
| 2.5 | 9.15 x108 |
| 3 | 2.43 x109 |
| 3.5 | 2.555 x109 |
| 4 | 2.7875 x109 |
| 4.5 | 2.225 x109 |

**8July (Fri)**

**1.** **Streak plate of Bl21 competent cell**

Ø   incubate at 37 oC

**2.** **transformation of Pwt: BBa\_E0044 (Green Fluorescent Protein deviated from jellyfish Aequeora Victoria wild-type GFP with plasmid pSB1A3, in 2011 kit plate 1 well 14G)**

Ø   10ul H20 added to well 14G

Ø   DNA concentration was measured: 85.1ug/ul

Ø   1ul of DHA was added to DH5a competent cell

Ø   2 tubes of competent cell was used, one being taken from other bb (labeled as DH5a T) and one being prepared by overselves (labeled as DH5a)

**Week 6** **Monday 11 July – Sunday 17 July**

**11 July (Mon)****1. iGEM plasmid stock check:**

|  |  |
| --- | --- |
| pSB1K3 | ~ 0ul |
| pSB1C3 | < 1ul |
| pSB1T3 | ~4-5ul |
| pSB1A3 | ~4-5ul |

Ø   Insufficient quantity

Ø   Bacterial amplification (plasmid+biobrick) of pSB1A3 (taken from iGEM kit plate 1, 1G)

Ø   Use competent cell prepared on 30June

Ø   Transformation: put pSB1A3 + biobrick into DH5a

Ø   Result refered to 13July

**12 July(Tue)****1. Nano drop**

|  |  |
| --- | --- |
| T7 + 1K3 | 42.5 ng/ul |
| HR + 1K3 | 13.5 ng/ul |

Ø   pSB1T3 labeled to be 25ng/ul

**1.** **Double digestion**

Ø   Incubated at 37 oC, 2hours

|  |  |
| --- | --- |
| H2O | 19.25ul |
| Buffer 2 | 2ul |
| BSA | 2.5ul |
| Eco R1 | 0.5ul |
| Spe 1 | 0.5ul |
| T71K3 | 0.25ul |
| TOTAL | 25ul |

|  |  |
| --- | --- |
| H2O | 14.25ul |
| Buffer 2 | 2.5ul |
| BSA | 0.25ul |
| Xbal 1 | 0.5ul |
| Pst 1 | 0.5ul |
| HR1K3 | 7ul |
| TOTAL | 25ul |

|  |  |
| --- | --- |
| H2O | 17.25ul |
| Buffer 2 | 2.5ul |
| BSA | 0.25ul |
| Eco R1 | 0.5ul |
| Pst 1 | 0.5ul |
| pSB1T3 | 4ul |
| TOTAL | 25ul |

Ø   Result: yield of product is too low

**2.** **Prepare T plate**

**3.** **Bacterial amplification of backbone + biobrick (Continue experiment on 11July)**

Ø   red colonies observed, planned to do Midi Prep on 13July

**4.** **1ml** **DH5α start culture solution is pipetted to each conical flasks**

Ø   The LB IPTG solution is contaminated, OD and cell count is too high and out of the normal range.

Ø   Frankie advised to use 500ml next time

Ø   Brian will check if the cell count machine works

Ø   Preparation of NaCl, KCl DH5α should include using pipette to draw all medium away to avoid bacteria killed by ice.

|  |  |  |  |
| --- | --- | --- | --- |
| Time(h)\OD600 of Solution | LB IPTG | 0.4M NaCl LB | 0.4M KCl LB |
| 0 | 0.180 | 0.049 | 0.025 |
| 0.5 | 0.234 | 0.021 | 0.020 |
| 1 | 0.301 | 0.041 | 0.024 |
| 1.5 | 0.377 | 0.047 | 0.055 |
| 2 | 0.606 | 0.127 | 0.114 |
| 2.5 | 0.744 | 0.209 | 0.212 |
| 3 | 0.916 | 0.324 | 0.331 |
| 3.5 | 1.125 | 0.472 | 0.449 |
| 4 | 1.289 | 0.673 | 0.725 |
| 4.5 | 1.432 | 0.881 | 0.886 |
| 5 | 1.533 | 10147 | 1.122 |
| 5.5 | 1.654 | 1.345 | 1.364 |

Ø   Cell density of DH5α, pET27b HR, LB IPTG with K

|  |  |
| --- | --- |
| Time(h) | Averaged cell density |
| 0 | 2.21x107 |
| 0.5 | 8.3 x108 |
| 1 | 7.9 x107 |
| 1.5 | 3.6 x108 |
| 2 | 3.035 x109 |

**13July (Wed)**

**1.** **Double digestion (repeat experiment of yesterday)**

Ø   Modify the volume to minimize the amount of insert(T7, HR)

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| Eco R1 | 0.5ul |
| Spe 1 | 0.5ul |
| T71K3 | 16.8ul |
| TOTAL | 20ul |

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| Xbal 1 | 0.5ul |
| Pst 1 | 0.5ul |
| HR1K3 | 16.8ul |
| TOTAL | 20ul |

|  |  |
| --- | --- |
| H2O | 12.8ul |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| Eco R1 | 0.5ul |
| Pst 1 | 0.5ul |
| pSB1T3 | 4ul |
| TOTAL | 20ul |

**2.** **Run gel to check cut products**

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| blank | HR+1K3 | Ladder | T7+1K3 | blank | pSB1T3 | blank | blank |

**3.** **Transformation of HR+1K3, T7+1K3 prepared on 28June**

Ø   plates spread with antibiotics K, incubate overnight

Ø   plates labeled as “HR+1K3 prepared on 28June” and “T71K3 prepared on 28June”

**4.** **Red colonies observed on plates prepared on 11July**

Ø   Miniprep tomorrow

**5.** **Gel check after double digestion and gel extraction**

Ø   Lane3: ladder

Ø   Lane6: T7 (13.1 ng/ul) ~200kb

Ø   Lane9: HR (25.5 ng/ul) ~800kb

Ø   Lane12: 1T3 (2.3ng/ul)

Ø   Gel result: weak banding

**6.** **Ligation**

|  |  |
| --- | --- |
| T4 ligase buffer | 2ul |
| T4 ligase | 1ul |
| T7 | 8ul |
| HR | 6ul |
| Vector (pSB1T3) | 3ul |
| TOTAL | 20ul |

**14July (Thu): no record**

**15July (Fri)**

**1.** **Transformation**

Ø   DH5a competent cell prepared on 30June

Ø   3 way ligation (T7- HR- 1T3)

**2.** **Inoculation**

Ø   Red colonies on plates prepared on 17July

Ø   2 centrifuge tubes prepared, labeled as red colony1 & 2

Ø   Transferred 250ul to 125mL LB (1:500 ratio)

Ø   Shake at 37 oC, 250rpm, overnight

Ø   Labeled as red col 1&2

**16July (Sat)**

**1.** **Mini prep**

Ø   DNA from red colony culture

Ø   4 tubes of DNA obtained

**2.** **Digestion**

|  |  |
| --- | --- |
| DNA | 10ul |
| 10X Buffer 3 | 2.5ul |
| 10X BSA | 2.5ul |
| EcoR1 | 0.6ul |
| Pst1 | 0.6ul |
| H2O | 8.8 |
| Total | 25ul |

Ø   Incubate at 37oC for 2hrs

**3.** **Pick colonies of 3-way assemblyDH5a**

Ø   Incubate at 37oC, 250rpm

**17 July (Sun)**

**1.** **Run gel and gel recovery of red colony**

**2.** **Mini prep, run gel and gel recovery of 3-way assembly**

**3.** **Run gel using digestion product prepared on 16/7 (red colony)**

**4.** **Mini prep DNA of bacteria culture (3-way DH5a)**

Ø   Total of 5 samples of 3-way 1T3 are made, each with 40ul

Ø   Stored at -20oC

Ø   10ul of each is used for digestion and run gel to check size

|  |  |
| --- | --- |
| Water | 11.25ul |
| DNA | 10ul |
| Buffer 3 | 2.5ul |
| EcoR1 | 0.5ul |
| Pst1 | 0.5ul |
| BSA | 0.25ul |
| Total | 25ul |

Ø   Incubate at 37oC for 2hrs

**5.** **Gel extraction of gel with 1T3 backbone from red conlony**

Ø   4 tubes of backbone are made

Ø   Store at -20oC

**6.** **Pick red colony of DH5a**

Ø   4tubes of LB snap cap

Ø   Incubate at 37oC, 25rpm

**7.** **Transfer to 125ml LB**

Ø   Shake at 37oC overnight, 250rpm

**8.** **Streak plate using red colony**

Ø   Plates with antibiotics A are used

Ø   Store in incubator

**Week 7** **Monday 18 July – Sunday 24 July**

**18 July (Mon)**

**1.** **Nanodrop**

|  |  |  |
| --- | --- | --- |
| 3-way | 260/280 | ng/ul |
| 1T3 (1) | 1.88 | 56.3 |
| 1T3 (2) | 1.91 | 71.5 |
| 1T3 (3) | 1.91 | 33.5 |
| 1T3 (4) | 1.96 | 20.8 |
| 1T3 (5) | 1.87 | 30.1 |
|  |  |  |
| 1T3 backbone (1) | 3.78 | 3.9 |
| 1T3 backbone (2) | 1.91 | 5.8 |
| 1T3 backbone (3) | 1.77 | 2.0 |
| 1T3 backbone (4) | 1.42 | 1.9 |

**2.** **Mini prep of DH5a with kit plate 1G (red colony)**

Ø   5 tubes are prepared labeled as 1G (1) to (5)

Ø   Store at -20oC

Ø   Nanodrop

|  |  |
| --- | --- |
|  | ng/ul |
| 1G (1) | 6.37 |
| 1G (2) | 0.04 |
| 1G (3) | 0.22 |
| 1G (4) | 4.48 |
| 1G (5) | 0.04 |

**3.** **Run gel (3-way 1T3)**

Ø   Show banding at ~4000-5000

**4.** **Gel recovery of 3-way 1T3**

**5.** **Double digestion of 3-way 1T3**

**6.** **Run gel**

Ø   No banding shown

**7.** **Restriction cut of 1G (red colony)**

**8.** **Run gel 1G (red colony)**

Ø   Band at ~1000 and ~3000-4000

**9.** **Transformation**

Plasmid: T71K3 and HR1T3

DH5a competent cells prepared on 30/6

**10.****Spread plates**

**11.****Prepared 5 conical flasks with the following components**

Ø   LB with 50μl IPTG + 100 μl Antibiotic K

Ø   LB of 0.4 M NaCl with 50 μl IPTG + 100 μl Antibiotic K

Ø   LB of 0.6 M NaCl with 50 μl IPTG + 100 μl Antibiotic K

Ø   LB of 0.4 M KCl with 50 μl IPTG + 100 μl Antibiotic K

Ø   LB of 0.6 M KCl with 50 μl IPTG + 100 μl Antibiotic K

**12.****Transformed 5** **ml DH5****α with pET27b start culture and put them into the incubator at 37****°C in the common room.**

**19 July (Tue)**

**1.** **Pick colonies that we spread yesterday**

Ø   T71K3 (with antibiotics K added)                  x2

Ø   HR1T3                                                                                   x2

**2.** **Inoculation at 37oC, 250rpm for 6hrs**

Ø   Failed

**3.** **Add 500** **μl DH5****α start culture into 5 respective conical flasks prepared on 18/7**

Ø   record the OD600 of 5 conical flasks and the results are recorded.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Time (h) | LB+IPTG | 0.4M NaCl +LB +IPTG | 0.6M NaCl +LB +IPTG | 0.4M KCl+ LB+ IPTG | 0.6M KCl + LB +IPTG |
| 0 | 0.008 | 0.013 | 0.013 | 0.011 | 0.004 |
| 0.5 | 0.023 | 0.021 | 0.021 | 0.027 | 0.017 |
| 1.0 | 0.025 | 0.013 | 0.015 | 0.009 | 0.005 |
| 1.5 | 0.049 | 0.026 | 0.010 | 0.012 | 0.005 |
| 2.0 | 0.083 | 0.017 | 0.011 | 0.013 | 0.003 |
| 2.5 | 0.209 | 0.033 | 0.015 | 0.045 | 0.010 |
| 3.0 | 0.382 | 0.107 | 0.058 | 0.107 | 0.054 |
| 3.5 | 0.567 | 0.234 | 0.082 | 0.177 | 0.062 |
| 4.0 | 0.783 | 0.329 | 0.126 | 0.288 | 0.128 |
| 4.5 | 0.973 | 0.485 | 0.165 | 0.432 | 0.206 |
| 5.0 | 1.214 | 0.673 | 0.258 | 0.662 | 0.252 |
| 5.5 | 1.454 | 0.906 | 0.386 | 0.845 | 0.336 |
| 6.0 | 1.554 | 1.132 | 0.450 | 1.078 | 0.457 |

![](http://samlo.t0.ru/igem/w701.png)

**4.** **Transformed 3 start cultures of DH5****α with pET27b with the total volume of 5ml with the following components**

Ø   5ml LB + 5 μl K

Ø   3ml LB + 2ml of 1M NaCl + 5 μl K (LB with 0.4M of NaCl)

Ø   2ml LB + 3ml of 1M NaCl + 5 μl K (LB with 0.6M of NaCl)

**20 July (Wed)**

**1.** **Pick colony**

Ø   HR1T3 X1

Ø   T71K3 X1

Ø   Red colony X3

Ø   All with antibiotics added

**2.** **Incubation**

Ø   37 oC, 250rpm, 6hours

**3.** **Transfer**

Ø   37 oC, 240rpm, overnight

**4.** **Prepared the autoclaved the 4 solutions**

Ø   500 ml LB                                                                  X2

Ø   500 ml LB constituting 1M NaCl       X1

Ø   500 ml LB constituting 1M KCl                     X1

**5.** **Prepared 5 conical with the following constitute**

Ø   LB with 50μl IPTG

Ø   LB of 0.4 M NaCl with 50 μl IPTG

Ø   LB of 0.6 M NaCl with 50 μl IPTG

Ø   LB of 0.4 M KCl with 50 μl IPTG

Ø   LB of 0.6 M KCl with 50 μl IPTG

**6.** **Transformed 5 DH5****α with pET27b start solution with different NaCl and KCl concentrations**

Ø   5ml LB + 5 μl antibiotic K

Ø   5ml of 0.4M NaCl + 5 μl antibiotic K

Ø   5ml of 0.6M NaCl + 5 μl antibiotic K

Ø   5ml of 0.4M KCl + 5 μl antibiotic K

Ø   5ml of 0.6M KCl + 5 μl antibiotic K

**21July (Thu)**

**1.** **Midi Prep of red colony (1G) & HR**

Ø   3 samples of 1G: 35.7ng/ul, 27ng/ul, 124ng/ul

Ø   1 sample of HR: 5.0ng/ul

**2.** **Digestion of 1G red colony using 1G(1) & 1G(3)**

Ø   1G(1)

|  |  |
| --- | --- |
| Buffer 3 | 2.5ul |
| 100X BSA | 0.25ul |
| Eco R1 | 0.5ul |
| Pst 1 | 0.5ul |
| DNA | 14ul (500ng) |
| H20 | 7.25ul |

Ø   1G(3)

|  |  |
| --- | --- |
| Buffer 3 | 2.5ul |
| 100X BSA | 0.25ul |
| Eco R1 | 0.5ul |
| Pst 1 | 0.5ul |
| DNA | 4ul (500ng) |
| H20 | 17.25ul |

Ø   Incubated at 37oC, 2hours

**3.** **Run gel using digestion products**

Ø   Band size: ~1000 and 2000-2500kb

**4.** **Could not measure the absorbance of LB of 0.6M NaCl, 0.4M KCl and 0.6M KCl because the start culture of DH5****α cannot grow properly. The absorbance of start culture of LB and LB of 0.4M NaCl prepared on 20/7/2011 without adding DH5****α were measured. Other than that, we also measured the absorbance of LB+ IPTG and LB of 0.4M NaCl+ LB + IPTG**

Ø   Result

|  |  |  |
| --- | --- | --- |
|  | LB + IPTG | LB of 0.4M of NaCl |
| Start culture absorbance (1ml) | 1.323 | 0.464 |

5.

|  |  |  |
| --- | --- | --- |
| Time (h) | LB+ IPTG | LB of 0.4M NaCl + IPTG |
| 0 | 0.021 | 0.014 |
| 0.5 | 0.026 | 0.024 |
| 1.0 | 0.045 | 0.033 |
| 1.5 | 0.043 | 0.036 |
| 2.0 | 0.133 | 0.064 |
| 2.5 | 0.245 | 0.105 |
| 3.0 | 0.359 | 0.159 |
| 3.5 | 0.555 | 0.228 |
| 4.0 | 0.720 | 0.303 |
| 4.5 | 0.927 | 0.466 |
| 5.0 | 0628 (2X) | 0.618 |
| 5.5 | 0.846(2X) | 0.815 |
| 6.0 | 0.899(2X) | 0.610(2X) |

![](http://samlo.t0.ru/igem/w702.png)

**22July (Fri)**

**1.** **Run gel (HR)**

**2.** **Transformation**

Ø   3A pSB1C3

Ø   5A pSB1K3

Ø   7A pSB1T3

Ø   T7 1K3

Ø   DH5a competent cells

**3.** **Spread plate**

Ø   3C plates of 3A-pSB1C3

Ø   4K plates of 5A-pSB1K3

Ø   4T plates of 7A-pSB1T3

Ø   3K plates of T7-1K3

**23July (Sat)**

**1.** **Digestion using 1G(3) prepared on 21July2011**

Ø   incubated at 37 oC, 2hours

Ø   product stored in 4 oC fridge with label 1G backbone 23h

**2.** **Pick colony & Inoculation**

Ø   using plates prepared on 22July

-          3A pSB1C3

-          5A pSB1K3

-          7A pSB1T3

-          T7-1K3

Ø   3 snap caps containing 5ml LB and corresponding antibiotics are prepared for each type of plate.

-          3A pSB1C3 23/7 莊

-          5A pSB1K3 23/7 莊

-          7A pSB1T3 23/7 莊

-          T7-1K3 23/7 莊

-          Snap caps are put in common shaker

**24July (Sun)**

**1.** **Mini prep using cultures prepared yesterday, 24 samples are made:**

Ø   Stored in -20 oC fridge

-          3A1C3 (1-8)

-          7A1T3 (1-4)

-          5A1K3 (1-4)

-          T71K3 (1-8)

****Week 8****Monday 25 July – Sunday 31 July**

**25 July (Mon)**

1.      **Restriction cut**

Ø   1G (1A3)

2.      **Run gel to extract backbone 1A3**

Ø   2 bands observed: ~1000 and ~2000-3000

3.      **Gel recovery**

4.      **Nanodrop****of 24 samples made on 24/7**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
|  | ng/ul |  | ng/ul |  | ng/ul |
| 3A1C3 (1) | 58.7 | 5A1K3 (1) | 48.0 | T71K3 (1) | 47.6 |
| 3A1C3 (2) | 42.8 | 5A1K3 (2) | 24.2 | T71K3 (2) | 46.8 |
| 3A1C3 (3) | 51.8 | 5A1K3 (3) | 12.1 | T71K3 (3) | 47.6 |
| 3A1C3 (4) | 58.1 | 5A1K3 (4) | 12.0 | T71K3 (4) | 33.9 |
| 3A1C3 (5) | 29.0 | 7A1T3 (1) | 35.2 | T71K3 (5) | 47.1 |
| 3A1C3 (6) | 28.6 | 7A1T3 (2) | 24.2 | T71K3 (6) | 43.6 |
| 3A1C3 (7) | 13.9 | 7A1T3 (3) | 27.6 | T71K3 (7) | 41.4 |
| 3A1C3 (8) | 34.9 | 7A1T3 (4) | 27.1 | T71K3 (8) | 31.9 |

5.      **Transformed competent DH5****α cells with vector pET27b HR and then spread them on K plate. The plate was then put into the incubator at 37****°C**

**26 July (Tue)**

**1.****Restriction cut using HR and T7**

|  |  |
| --- | --- |
| Buffer 3 | 2.5ul |
| BSA | 0.25ul |
| EcoR1 | 0.5ul |
| Pst1 | 0.5ul |
| DNA (35.3ng/ul) | 14.25ul |
| H2O | 7.0ul |
| Total | 25ul |

|  |  |
| --- | --- |
| Buffer 3 | 2.5ul |
| BSA | 0.25ul |
| EcoR1 | 0.5ul |
| Pst1 | 0.5ul |
| DNA (180ng/ul) | 21.25ul |
| Total | 25ul |

Ø   Incubate at 37oC for 2hrs

**2.****Run gel**

Ø   Result:     HR: ~800bp

T7: 200bp

**3.****Gel recovery**

**4.****3-way assembly**

|  |  |
| --- | --- |
| H2O | 11ul |
| 10X T4 DNA ligase buffer | 2ul |
| T4 DNA ligase | 1ul |
| HR | 2ul |
| T7 | 2ul |
| 1A3 | 2ul |
| Total | 20ul |

**5.****Prepared 5 conical flasks**

Ø   LB with 50μl IPTG

Ø   LB of 0.4 M NaCl with 50 μl IPTG

Ø   LB of 0.6 M NaCl with 50 μl IPTG

Ø   LB of 0.4 M KCl with 50 μl IPTG

Ø   LB of 0.6 M KCl with 50 μl IPTG

**6.****5 start cultures were also prepared**

Ø   5ml LB + 5 μl antibiotic K

Ø   5ml of 0.4M NaCl + 5 μl antibiotic K

Ø   5ml of 0.6M NaCl + 5 μl antibiotic K

Ø   5ml of 0.4M KCl + 5 μl antibiotic K

Ø   5ml of 0.6M KCl + 5 μl antibiotic K

**27 July (Wed)**

**1.****Transform terminator (iGEM plate2 24C) into DH5a**

**2.****Restriction cut**

Ø   HR1K3 and HR-T71A3 (3-way assembly) by EcoR1 and Pst1

**3.****Run gel**

Ø   Ladder mix together, cannot see size

**4.****Spread plates**

Ø   6 K-plates of 24C terminator

**5.****Measured the absorbance of the 5 conical flasks prepared on the 26/7 by cell counting machine with the following result**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Start culture | LB | KCl 0.4M | NaCl 0.4M | KCl 0.6M | NaCl 0.6M |
| First measurement (4X) | 0.430 | 0.674 | 0.410 | 0.380 |  |
| Second measurement  (4X) | 0.401 | 0.636 | 0.431 | 0.488 | 0.348 |

Ø   Preciously, we use the qViro cell counter

Result

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Time (h)/ culture | LB | KCl 0.4M | NaCl 0.4M | KCl 0.6M | NaCl 0.6M |
| 0 | 0.023 | 0.046 | 0.027 | 0.022 | 0.025 |
| 0.5 | 0.025 | 0.038 | 0.029 | 0.029 | 0.027 |
| 1.0 | 0.036 | 0.032 | 0.026 | 0.039 | 0.048 |
| 1.5 | 0.078 | 0.038 | 0.035 | 0.043 | 0.041 |
| 2.0 | 0.142 | 0.104 | 0.057 | 0.038 | 0.040 |
| 2.5 | 0.295 | 0.154 | 0.113 | 0.053 | 0.053 |
| 3.0 | 0.437 | 0.287 | 0.179 | 0.077 | 0.067 |
| 3.5 | 0.601 | 0.408 | 0.270 | 0.126 | 0.104 |
| 4.0 | 0.796 | 0.580 | 0.373 | 0.162 | 0.146 |

**![](http://samlo.t0.ru/igem/stock/image006.png)**

**28 July (Thur)**

**1.****Collect plates from 27/7, store at 4oC**

**2.****Run gel**

Ø   3-way assemblt plasmid made yesterday

Ø   No bands shown

**3.****Restriction cut of pSB1A3**

|  |  |
| --- | --- |
| Buffer 2 | 2.5ul |
| BSA | 0.25ul |
| EcoR1 | 0.5ul |
| Pst1 | 0.5ul |
| H2O | 16ul |
| DNA | 5.25ul |
| Total | 25ul |

Ø   37oC for 2hrs

**4.****Run gel**

Ø   No bands shown

**5.****Measured the absorbance change over time of the flask LB IPTG (100ml) with DH5****α transformed with pET27b measured and counted the cells in CSLB G12**

Result

|  |  |  |
| --- | --- | --- |
| Time (h) | OD600 | Cell Count (particle per ml) |
| 0 | -0.004 | 4.5 X 108 |
| 0.5 | -0.002 | 5.2 X 108 |
| 1.0 | ±0.000 | 6.7 X 108 |
| 1.5 | +0.002 | 8.7 X 109 |
| 2.0 | +0.007 | 1.8 X 109 |
| 2.5 | +0.026 | 1.8 X 109 |
| 3.0 | +0.041 | 3.1 X 109 |
| 3.5 | +0.061 | 4.8 X 109 |

![](http://samlo.t0.ru/igem/stock/image008.png)

![](http://samlo.t0.ru/igem/stock/image010.png)

**29July (Fri)**

**1.****Run gel**

Ø   **45min, 120V**

Ø   **6X loading dye: 4ul, DNA: 20ul**

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **Lane** | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
|  | ladder |  | 1G cut | T7 cut | HR cut |  |  |  |

Ø   Result
- lane 3: 1000-1500bp & 2000-3000bp- lane 4: 100-200bp
- lane 5: 500-1000bp

**2.****Gel clean of T7, HR, 1G**

Ø   Labeled: “T7 28/7 Mo”, “HR 28/7 Mo”, “1G 29/7 Mo”

Ø   Stored in 4oC fridge

**3.****Ligation using T7, HR, 1G**

Ø   Incubated at 16oC

|  |  |
| --- | --- |
| HR | 7ul |
| T7 | 7ul |
| 1G | 2ul |
| Ligation buffer | 2ul |
| T4 ligase | 1ul |
| H2O | 1ul |
| TOTAL | 20ul |

**4.****100mM MQAE solution and alinquoted into 1.5ml black microcentrifuge tube with 500****μl each stored at -20****°C. 5****μl MQAE and 95****μl salt solution were added to microplate and microplatereaders was used in SC 127.**

Ø   MQAE

|  |  |
| --- | --- |
| Mass (mg) | 100 |
| Molar mass (g mol-1 ) | 326.1893 |
| Concentration (mM) | 100 |
| Volume made (ml) | 3.066 |

Ø   Concentration of the salt solution with 2-fold dilution

i.                100mM NaCl

ii.                50mM NaCl

iii.                25mM NaCl

iv.                12.5mM NaCl

v.                6.25mM NaCl

vi.                3.125mM NaCl

Ø   Final concentration of MQAE in microplate reader is 5mM

**30July (Sat)**

**1.****PCR amplification of T7 & HR**

Ø   Labeled as T7 1, T7 2, HR

**31July (Sun)**

**1.****Run gel using PCR product**

**2.****Gel recovery**

Ø   1 tube of HR, 1 tube of T7, each of 30ul

**3.****Digestion**

Ø   1G, 3A1C3, 5A1K3, 7A1T3

Ø   Use products to run gel, obtain backbone

**4.****Ligation**

Ø   (HR, T7) & (1C3, 1K3, 1T3 backbone)

Ø   4 samples: T71K3, T71T3, HR1K3, HR1C3

Ø   Incubated at 16oC, overnight

**5.****Transformation**

Ø   use competent cell prepared on 30/6

Ø   4 samples: 3A1C3, 3A1T3, 5A1K3, 1G

Ø   Spread plates: using A,K,C,T plates, each type 2 plates**

**Week 9****Monday 1 Aug – Sunday 7 Aug**

**1****Aug****(Mon****)**

**1. PCR amplification of T7 and HR**

**2. PCR purification of T7 and HR**

**3. Transformation**

Ø   1G, 3A, 5A, 7A

-Used DH5a competent cells

-2 sets are prepared

Ø   HR1C3, HR1K3, T71T3, T71K3

-Used DH5a competent cells

-2 sets are prepared

Ø   chloride sensor

-Used DH5a competent cells

-1 tube is prepared

**4. Spread plate**

Ø   One plate is used for each tube of transformed competent cells

Ø   All plates stored in incubator at 37oC

**5. Run gel to check size**

Ø   Result:     HR: ~800

T7: ~100-200

**6.****Prepared 1 flask comprising 100ml LB, 50****μl IPTG and 100****μl antibiotic K and also 1 DH5****α with pET27b HR start culture**

**2****Aug (Tue****)**

**1.****Inoculation**

Ø  HR1K3 (failed)

Ø  HR1C3

Ø  T71K3  (failed)

Ø  T71T3

Ø  3A

Ø  5A

Ø  7A

Ø  1G

Ø  Chloride sensor   (failed)

**2.****Restriction cut**

Ø   HR, T7, 1T3

**3.****Ligation**

Ø   HR1T3 and T71T3

Ø   Store at -16oC overnight

**4.****Transfer to 125ml conical flask for MIDI Pre**

**5.****Transformation**

Ø   24C terminator and DH5a used

Ø   each spread on 2 plates

**6.****Prepare agar plate**

Ø   C plates           x18

Ø   A plates           x20

Ø   K plates           x21

**7.****Absorbance of the start culture was measured and the cells were counted**

|  |  |  |
| --- | --- | --- |
| Time (h) | OD600 | Cell count (cell/ml) |
| 0 | 0.000 | 4 X 108 |
| 0.5 | 0.003 | 1.4 X 1010 |
| 1.0 | 0.008 | 8.6 X 107 |
| 1.5 | 0.022 | 3.3 X 108 |
| 2.0 | 0.142 | 3 X 108 |
| 2.5 | 0.203 | 7 X 108 |
| 3.0 | 0.263 | / |

Result

![](http://samlo.t0.ru/igem/stock/image012.png)

Ø   Brian suggested that the abnormality of the cell count data might be due to the growing size of the bacteria. At 3.0 hour, signal could not be obtained as well as the reason.

**3****Aug****(Wed****)**

**1.****Mini prep**

Ø  3A

Ø  5A

Ø  7A

Ø  T71T3

Ø  Cl sensor

**2.****3****-way assembly**

|  |  |  |
| --- | --- | --- |
|  | Ng/ul | 260/280 |
| 3-way | 12.8 | 1.93 |
| Cl sensor | 1.2 | 1.85 |
| T71T3 | 13.0 | 1.93 |
| HR1C3 | 103.0 (15.9) | 1.33 |
| 5A | 35.3 | 1.87 |
| 7A | 24.2 | 1.90 |

**3.****Concentration of the NaCl solution was prepared as follow by serial dilution**

Ø  1000mM NaCl

Ø  500mM NaCl

Ø  100mM NaCl

Ø  50mM NaCl

Ø  25mM NaCl

Ø  12.5mM NaCl

Ø  6.25mM NaCl

Ø  3.125mM NaCl

**4.****Concentration of MQAE was also prepared as follow by serial dilution**

Ø   100mM MQAE

Ø   10mM MQAE

Ø   1mM MQAE

**5.****95****μl of NaCl solutions and 5****μl****of MQAE solutions were added into the microplate in the position as shown below.**

![](http://samlo.t0.ru/igem/stock/image013.png)

As a result, the final MQAE concentration

Ø   A1-A4,B1-B4, C1-C4, D1-D4, E1-E4, F1-F4, H1-H4 are 5mM

Ø   A5-A8, B5-B8, C5-C8, D5-D8, E5-E8, F5-F8, H5-H8 are 0.5mM

Ø   A9-A12, B9-B12, C9-C12, D9-D12, E9-E12, F9-F12, H9-H12 are 0.05mM

Ø   The plate with aluminum foil was wrapped and put in the incubator at 37°C for 1 hour and measured emission by microplate reader

Ø   Result

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 5480 | 5633 | 5428 | 5554 | 3013 | 3267 | 3058 | 3209 | 1249 | 1335 | 1341 | 1267 |
| 8990 | 9199 | 9483 | 9446 | 5235 | 5222 | 5241 | 4649 | 1545 | 1646 | 1634 | 1630 |
| 763 | 21493 | 21870 | 23048 | 11411 | 11549 | 11711 | 10767 | 2564 | 2513 | 2549 | 2413 |
| 25542 | 29517 | 27811 | 29312 | 14561 | 14986 | 14392 | 14646 | 3056 | 3018 | 2848 | 2839 |
| 30141 | 32470 | 33457 | 27985 | 17182 | 17219 | 14902 | 19335 | 3351 | 3318 | 2974 | 2931 |
| 37055 | 36110 | 39855 | 35193 | 18985 | 18717 | 18398 | 16975 | 3565 | 3349 | 3216 | 3366 |
| 34258 | 36477 | 37255 | 35236 | 18320 | 18588 | 18778 | 6182 | 3425 | 3211 | 3424 | 3265 |
| 36893 | 37123 | 37393 | 35904 | 13574 | 25312 | 19447 | 1334 | 3386 | 3529 | 3191 | 3364 |

|  |
| --- |
| A: 1000mM NaCl |
| B: 500mM NaCl |
| C: 100mM NaCl |
| D: 50mM NaCl |
| E: 25mM NaCl |
| F: 12.5mM NaCl |
| G: 6.25mM NaCl |
| H: 3.125mM NaCl |

**4****Aug (Thu****)**

**1.  Mini prep**

|  |  |  |
| --- | --- | --- |
|  | ng/ul | 260/280 |
| 24C (1) | 44.1 | 1.77 |
| 24C (2) | 59.7 | 1.76 |
| T71K3 (1) | --- | --- |
| T71K3 (2) | 41.7 | 1.87 |
| HR1K3 (1) | 18.5 | 1.92 |
| HR1K3 (2) | 29.5 | 1.78 |
| 3A (1) | --- | --- |
| 3A (2) | 50.7 | 1.88 |
| 5A (1) | 44.4 | 1.85 |
| 5A (2) | --- | --- |
| 1G (1) | 142.9 | 1.86 |
| 1G (2) | 52.4 | 1.89 |
| 7A | 43.5 | 1.86 |

Ø   3A (1) is red while 3A (2) is milky

Ø   1G (1) is red while 1G (2) is milky

**2.****Double digestion**

Ø   T71K3

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.5ul |
| Spe1 | 0.5ul |
| T71K3 (prepared on 4/8) | 16.8ul |
| Total | 20ul |

Ø   HR1C3

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.5ul |
| Pst 1 | 0.5ul |
| HR1C3 (prepared on 3/8) | 16.8ul |
| Total | 20ul |

Ø   7A

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.5ul |
| Pst 1 | 0.5ul |
| 7A (prepared on 3/8) | 4ul |
| H2O | 12.8ul |
| Total | 20ul |

-37oC, 2hours

**3.****3-way ligation**

|  |  |
| --- | --- |
| T4 ligase buffer | 2ul |
| T4 ligase | 1ul |
| T7 | 10ul |
| HR | 4ul |
| Vector (7A) | 3ul |
| Total | 20ul |

**4.****Inoculation**

Ø   Pick colony of white from T71T3, HR1T3

**5****Aug (Fri****)**

**1.****Nanodrop**

|  |  |  |
| --- | --- | --- |
| Samples prepared on 4/8 | ng/ul | 260/280 |
| HR1T3 (1) | 10.3 | 1.98 |
| HR1T3 (2) | 12.2 | 1.91 |
| T71T3 (1) | 10.5 | 1.82 |
| T71T3 (2) | 12.4 | 1.86 |

**2.****Transformation**

Ø   used DH5a competent cells

Ø   only 1 set is prepared using 3-way ligation products

Ø   Plate was spreaded after transformation using T plates

(plates labeled: 5/8transformation, 3A ligation)

Ø   store st 37oC

**3.****Pick colonies**

Ø   pick from T71T3

Ø   3 colonies are picked

Ø   inoculate for 12-16hrs

Ø   3 snapcap tubes, 37oC, 250rpm

**7****Aug****(Sun****)**

**1.****inoculation form plates HR-T7-1T3 prepared on 6/8**

**2.****pick 3 colonies to snap-cap tubes with 6ml LB, then shake at 250rpm, 37oC for 12-16 hrs**

**3.****Prepared and inoculated four tubes of the start culture with the volume of 10ml , which are 0.4M NaCl LB with light, 0.4M NaCl LB without light , LB with light, and LB without light. But we didn’t add IPTG.**

**4.****Measured OD 600 of four start culture**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | 0.4M NaCl LB with light | 0.4M NaCl LB without light | LB with light | LB without light |
| OD600 (2X) | 0.230 | 0.401 | 0.388 | 0.690 |

**5.****Spin four tubes of start culture separately to remove the LB/NaCl LB then store in -80****°C refrigerator.**

**6.****Prepared four tubes of start culture with the total volume of 10 ml, which 0.4M NaCl LB with light, 0.4M NaCl LB without light, LB with light and LB without light and inoculated them overnight 5****μl of 1M IPTG was added**

****Week 10****Monday 8 Aug – Sunday 14 Aug**

**8****Aug (Mon****)**

**1.****Mini prep 3-way ligation products prepared on 7/8**

Result

|  |  |  |
| --- | --- | --- |
|  | ng/ul | 260/280 |
| 3-way ligation | 11.5 | 1.83 |
| HR-T7-1T3 (1) | 13.9 | 1.86 |
| HR-T7-1T3 (2) | 13.2 | 1.91 |
| T71T3 (1), prepared on 6/8 | 29.4 | 1.52 |
| T71T3 (2) , prepared on 6/8 | 7.3 | 1.69 |
| T71T3 (3) , prepared on 6/8 | 5.4 | 1.74 |

Ø   DNA ladder used is from Fermentas GeneRuler 1kb DNA, #SM1331

**2.****Run gel**

**3.****Double digestion**

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.5ul |
| Spe 1 | 0.5ul |
| T71T3 (prepared on 6/8) | 16.8ul |
| Total | 20ul |

|  |  |
| --- | --- |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| XBa 1 | 0.5ul |
| Pst 1 | 0.5ul |
| HR1K3 (prepared on 4/8) | 16.8ul |
| Total | 20ul |
| Buffer 2 | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.5ul |
| Pst 1 | 0.5ul |
| 3A (prepared on 4/8) | 2ul |
| H2O | 14.8ul |
| Total | 20ul |

Ø   37oC, 2hrs

**4.****Ligation**

|  |  |
| --- | --- |
| T4 ligase buffer | 2ul |
| T4 ligase | 1ul |
| T7 | 7ul |
| HR | 7ul |
| Vector pSB1C3 from 3-way | 3ul |
| Total | 20ul |

Ø   16oC overnight

**5.****Measured OD600of the four start cultures of 10ml of IPTG made on 7/8 then spin four tubes of start culture separately to remove the LB/NaCl LB**

Ø   Put in the -80°C refrigerator for storage.

**6.****Prepared again four tubes of 10ml of start culture**

Ø   LB of 0.4M NaCl with light

Ø   LB of 0.4M NaCl without light

Ø   LB with light

Ø   LB without light

Ø   Inoculated them with the addition of 5μl of 1M IPTG.

**7.****Measured OD600 of the four start cultures.**

Result:

OD600of the four start cultures of 10ml of IPTG made on 7-8-2011

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | LB of 0.4M NaClwith light | LB of 0.4M NaClwithout light | LB with light | LB without light |
| OD600(2X) | 0.328 | 0.503 | 0.875 | 0.934 |

OD600 of the four start cultures were measured.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | LB of 0.4M NaClwith light | LB of 0.4M NaClwithout light | LB with light | LB without light |
| OD600 (2X) | 0.357 | 0.526 | 0.390 | 0.627 |

Ø   Spin four tubes to remove the LB/LB NaCl

Ø   Store in -80°C refrigerator.

**9****Aug****(Tue****)**

**1.****Transformation of 3-way ligation clone prepared on 8/8**

**2.****Transformation of T71T3 mini prep product prepared on 6/8**

**3.****Transformation of 3-way ligation prepared on 8/8**

Ø   All experiment were done following the standard transformation protocol, except that for: 1. 2ul added, 2. 2ul added, 3. 5ul added

**4.****Spread plates with antibiotics C, T respectively**

**5.****Pick colony from T71T3, HR1T3, 3-way ligation plates from refrigerator and then inoculate overnight**

**6.****We unfortunately make a mistake that because the OD was measured and spinned all of the solution, whereas the start solution should be spinned with accordance with the volume calculated from the measurement of the OD. Therefore, the whole experiment needs to be redone.**

**10****Aug****(Wed****)**

**1.****Mini prep of T71T3, HR1T3, 3-way ligation from 9/8**

|  |  |  |
| --- | --- | --- |
|  | ng/ul | 260/280 |
| T71T3 (1) | 111.0 | 1.84 |
| T71T3 (2) | 109.9 | 1.85 |
| T71T3 (3) | 147.9 | 1.90 |
| HR1T3 (1) | 103.0 | 1.89 |
| HR1T3 (2) | 98.9 | 1.85 |
| HR1T3 (3) | 107.6 | 1.84 |
| 3A (1) | 101.2 | .182 |
| 3A (2) | 123.6 | 1.88 |
| 3A (3) | 210.0 | 1.87 |

Ø   all are using 50ul H2O to elute al the final step, except that all tubes of 3 had been added 30ul to increase concentration

**2.****inoculation**

Pick 3 colonies from 3 plates respectively, totally of 9, from

Ø  3-way ligation(8/8) after mini prep done on 9/8

Ø  3-way ligation(8/8) done on 9/8

Ø  T71T3 (6/8) done on 9/8

Labeled as follows:

|  |
| --- |
| 3-way post miniprep (1) |
| 3-way post miniprep (2) |
| 3-way post miniprep (3) |
| 3-way (1) |
| 3-way (2) |
| 3-way (3) |
| T71T3 (1) |
| T71T3 (2) |
| T71T3 (3) |

**3.****Inoculated 4 DH5****α with pET27b HR start cultures namely,**

Ø   LB + IPTG +K with light

Ø   LB + IPTG +K without light wrapped with aluminum foil

Ø   LB + 0.4M NaCl + IPTG +K with light

Ø   LB  + 0.4M NaCl + IPTG +K without light wrapped with aluminum foil

**11 Aug (Thu)**

**1.****Miniprep****of 9 tubes prepared on 10/8**

|  |  |  |
| --- | --- | --- |
| **Tube** | **Concentration (ug/ul)** | **260/280** |
| 3A Post mini prep (1) | 96.1 | 1.91 |
| 3A Post mini prep (2) | 41.6 | 1.96 |
| 3A Post mini prep (3) | 100.5 | 1.85 |
| 3A (1) | 104.7 | 1.85 |
| 3A (2) | 68.8 | 1.87 |
| 3A (3) | 74.7 | 1.96 |
| T71T3 (1) | 87.3 | 1.79 |
| T71T3 (2) | 115.8 | 1.85 |
| T71T3 (3) | 135.9 | 1.91 |

Ø   all no. 3 tubes are eluted with 30ul 60oC water to increase yield and concentration

**2.****Transform plate 2 24C terminator**

Ø   use competent cell made on 30/6

Ø   24C terminator mini-prep on 4/8 by Eric

Ø   Plate stored at 37oC incubator, overnight

Ø   Remaining transformed 24C terminator stored at -20oC,
labeled as “ transf. 24C, 11/8,ii)

**3.****Sequencing**

Ø   9 tubes prepared on 10/8, 9 tubes prepared on 11/8

**4.****Measured the OD600 of the 4 start cultures prepared on 10/8**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | LB of 0.4M NaClwith light | LB of 0.4M NaClwithout light | LB with light | LB without light |
| OD600 (2X) | 0.266 | 0.267 | 0.302 | 0.286 |

**5.****10% SDS was diluted into 2% SDS by adding the autoclaved 40ml of distilled water and 10ml of 10% SDS based on the insensitivity of SDS**

**6.****Measure the OD600 of the second set without IPTG**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | LB of 0.4M NaClwith light | LB of 0.4M NaClwithout light | LB with light | LB without light |
| OD600 (2X) | 0.094 | 0.096 | 0.350 | 0.355 |
| Volume to bespinned (ml) | \*All | \*All | 4.5 | 4.5 |

All volume to be spinned because the OD of NaCl sets are too small so the total volumes are still inadequate. As a result, a back up set without IPTG by inoculation was prepared and collected on 12-8-2011. It was found that the DH5α plate has been used up for cline picking so later transformation and plate spreading is need. Besides, it was found that there is only little amount of 1000K anti-biotic too.

**12 Aug (Fri)**

**1.****Inoculation**

Ø   pick 3 colonies from plate “24C terminator transformed 11Aug”

**2.****Measured the OD600 of the back up set of bacteria.**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | LB of 0.4M NaClwith light | LB of 0.4M NaClwithout light | LB with light | LB without light |
| OD600 (2X) | 0.459 | 0.290 | 0.768 | 0.847 |
| Volume to bespinned (ml) | 3.5 | 5.5 | 2 | 1.9 |

**3.****Pellets after spinning down were stored in -80°C refrigerator**

Ø   2% of 100μl of SDS was used to lyse the cells in 99°C for 5 minutes

Ø   Serial dilution of the bacteria in 2X, 4X, 8X and 16X and of salt like previously done on 3/8 were done

Ø   The MQAE and the solutions were added into the microplate and incubated for an hour

**4.****Measured the MQAE fluorescence with microplate reader**

Ø   The data were abnormal and Frankie deduced that it may be due to the bubbles of 2% SDS

**13 Aug (Sat)**

**1.****Miniprep****of 24C terminator prepared on 12/8**

Ø   3 tubes are labeled as “24C (1) 13/8”, “24C (2) 13/8”, “24C (3) 13/8”**

**Week 11****Monday 15 Aug – Sunday 21 Aug**

**15 Aug (Mon)**

**1.****Transformed pET27bHR into DH5****α and spread on a K plate**

**16 Aug (Tue)**

**1.****Inoculated LB IPTF, 0.4M NaCl IPTG, 0.4M NaCl IPTG with light, 0.4M KCL IPTG and 0.4M KCl IPTG without light with DH5****α pET27b**

**17 Aug (Wed)**

**1.****Measured the OD 600 of the back up set of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600  (2X) | 0.857 | 0.724 | 0.733 | 0.316 | 0.660 |
| Volume to bespinned down (ml) | 1.762 | 2.086 | 2.060 | 4.778 | 2.288 |

**2.****2% of SDS was spinned to lyse the cells in 99****°C for 5 minutes with 100****μl each, Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out. The MQAE and solutions were added into the microplate and inoculated for an hour.**

**3.****Measured the MQAE fluorescence with microplate reader**

Ø   The data was also abnormal and Frankie deduced that it may be also due to the bubbles of the 2% SDS.

**4.****Inoculated the following tubes with DH5****α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 5μ of IPTG)

**18 Aug (Thu)**

**1.****Run gel with PCR products (T7, HR)**

Ø   Result: HR did not show band with 800 bp

**2.****Run gel using PCR product (T7)**

Ø   Result: the samples show unclear result

**3.****PCR with T7 & HR**

**4.****PCR purification of PCR product of step 3**

**![](http://samlo.t0.ru/igem/stock/image014.jpg)**

**5.****Measured the OD600 of the back up set of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.862 | 0.242 | 0.751 | 0.797 | 0.252 |
| Volume to bespinned down (ml) | 2.8 | 10 | 3.2 | 3.0 | 9.6 |

**6.****2% of SDS was spin to lyses the cells in 99°C for 5 minutes with 100μl each, Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out. The MQAE and solutions were added into the microplate and inoculated for an hour.**

**7.****MQAE fluorescence was measured with microplate reader**

Ø   The data was abnormal and may be due to the bubbles of the 2% SDS.

**19Aug (Fri)**

**1.****Run gel to check size of PCR amplified T7 & HR prepared on 18/08**

7.      HR: result failed, at ~100bp

8.      T7: result ok, at 100-200bp

![](http://samlo.t0.ru/igem/stock/image015.jpg)

![](http://samlo.t0.ru/igem/stock/image016.jpg)

**20Aug (Sat)**

**1.****Inoculation of 3 plate prepared last night**

Ø   6 tubes prepared

Ø   Abandoned because MidiPrep cannot be done on Sunday, will do miniprep instead)

**2.****PCR with HR templates (2tubes)**

**3.****Restriction vut T7 prepared on 18/8 & backbone 5A1C3**

Ø   1tube of T7

|  |  |
| --- | --- |
| Buffer | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.4ul |
| Pst1 | 0.4ul |
| T7 | 15ul |
| H20 | 2ul |
| TOTAL | 20ul |

Ø   2tubes of backbone

|  |  |
| --- | --- |
| Buffer | 2ul |
| BSA | 0.2ul |
| EcoR1 | 0.4ul |
| Pst1 | 0.4ul |
| backbone | 10ul |
| H20 | 7ul |
| TOTAL | 20ul |

**4.****PCR purification of PCR product HR (2tubes) (continued step 2)**

**5.****Run gel to check size of HR purified product**

Ø   Run 50ul of each HR and do gel recovery

Ø   Each lane of HR with 25ul

Ø   Result: size correct, ~800bp

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Lane | 1 | 2 | 3 | 4 | 5 | 6 |
|  | --- | 100bp ladder | HR-1 | HR-1 | HR-2 | HR-2 |

**6.****Ligation of T7 & 1C3, overnight**

Ø   3 tubes

|  |  |
| --- | --- |
| T4 ligase buffer | 2ul |
| T4 ligase | 1ul |
| T7 | 3ul |
| 1C3 | 14ul |
| TOTAL | 20ul |

**7.****Inoculation of 24C terminator DH5a**

**21 Aug (Sun)**

**1.****Miniprep****6 tubes of 24C terminator**

**2.****Transformation:**

Ø   T71C3 (1) prepared on 20/8

Ø   1G1A3

Ø   3A1C3

Ø   5A1K3

Ø   7A1T3

Ø   Spread plate:
- white colonies: T71C3
- red colonies: 1G1A3, 3A1C3, 5A1K3, 7A1T3

**3.****Restriction cut of HR and backbone 1C3**

Ø   HR: 10ul

Ø   Backbone X2: total 40ul

**4.****Ligation of HR & 1C3, overnight**

Ø   3 tubes labeled as “HR1C3(1-3)”

**5.****Inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 5μ of IPTG)

****Week 12****Monday 22 Aug – Sunday 28 Aug**

**22 Aug (Mon)**

**1.****Inoculation**

Ø   37oC, 240rpm

Ø   1G1A3, 7A1T3 àwhite colonies

Ø   3A1C3, 5A1K3àred colonies

Ø   Did not pick T71C3

**2.****Nanodrop****of 24C (X6) & HR (X3)**

|  |  |  |
| --- | --- | --- |
|  | ug/ul | 260/280 |
| 24C (1) | 10.5 | 1.72 |
| 24C (2) | 7.9 | 1.78 |
| 24C (3) | 8.2 | 1.73 |
| 24C (4) | 7.9 | 1.59 |
| 24C (5) | 11.7 | 1.54 |
| 24C (6) | 26.7 | 1.37 |
| HR (1) | 6 | 1.67 |
| HR (2) | 4.9 | 1.80 |
| HR (3) | 8 | 1.07 |

**3.****Transformation**

**4.****Transfer of 1G1A3, 7A1T3, 3A1C3, 5A1K3**

Ø   each type 2 tubes

**5.****inoculation of transferred product**

**6.****Measure OD600 of the back up set of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.303 | 0.350 | 0.637 | 0.689 | 0.269 |
| Volume to bespinned down (ml) | 8 | 6.9 | 3.8 | 3.5 | 9 |

![](http://samlo.t0.ru/igem/stock/image018.png)

![](http://samlo.t0.ru/igem/stock/image020.png)

**7.****2% of SDS was spin to lyses the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out

Ø   95μl of each concentration was added into each well

Ø   5μl of MQAE was added into each well afterwards

Ø   A duplicate was made. Sample prepared by Frankie and 5 μl of MQAE were added with the same volume

**8.****Measured the MQAE fluorescence with microplate reader**

Ø   Result of 0.4M NaCl IPTG without light lay in the range of our standards

**9.****Inoculated the following tubes with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 5μ of IPTG)

**23 Aug (Tue)**

**1.****midiprep****3A1C3, 5A1K3**

**2.****transform 24C, chloride sensor, 1G1A3, 7A1T3,T71C3 with DH5a cells**

Ø   spread the above to agar plates

Ø   each 2 plates

**3.****pick colonies and inoculation of the above**

Ø   result: 1G1A3 & chloride sensor are too dense, others are ok

**4.****Measured the OD600 of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.923 | 0.875 | 0.318 | 0.396 | 0.706 |
| Volume to bespinned down (ml) | 2.62 | 2.77 | 7.6 | 6.11 | 3.43 |

![](http://samlo.t0.ru/igem/stock/image022.png)

![](http://samlo.t0.ru/igem/stock/image024.png)

**5.****2% of SDS was spinned to lyse the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out

Ø   95μl of each concentration was added into each well

Ø   A duplicate was made. Sample prepared by Frankie and 5 μl of MQAE were added with the same volume.

Ø   MQAE fluorescence was measured by microplate reader and the results were sent to Frankie.

**6.****Routinely, the following tubes are inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 5μ of IPTG)

**24 Aug (Wed)**

**1.****miniprep****HR1C3**

Ø   Result:

|  |  |  |
| --- | --- | --- |
| **sample** | **Conc. (ng/ul)** | **260/280** |
| HR1C3 (1) | 30.5 | 1.74 |
| (2) | 24.7 | 1.81 |
| (3) | 19.9 | 1.94 |
| (4) | 22.1 | 1.97 |
| (5) | 28.3 | 1.80 |
| (6) | 50.0, 34.8, 21.0, 24.8, 25.3 | 1.94, 1.77, 2.25, 1.97, 1.91 |
| (7) | 42.1, 29.5 | 1.91, 1.92 |
| (8) | 24.6 | 1.78 |
| (9) | 31.4, 25.8 | 1.80, 1.91 |
| (10) | 18.9 | 1.91 |
| (11) | 19.2 | 1.99 |
| (12) | 48.2, 43.1, 45.8 | 1.88, 1.99, 1.92 |

**2.****transform 1ul chloride sensor to 20ul DH5a competent cells**

**3.****Measured the OD600 of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.667 | 0.148 | 0.107 | 0.178 | 0.181 |
| Volume to bespinned down (ml) | 1.81 | 9.5 | 9.5 | 9.5 | 9.5 |

**4.****Then we use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the well

Ø   95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

**5.****Measured the MQAE fluorescence by microplate reader.**

**6.****Following tubes are inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

**25 Aug (Thu)**

**1.****M****easured the OD600 of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.851 | 0.718 | 0.767 | 0.322 | 0.717 |
| Volume to bespinned down (ml) | 3.4 | 4.0 | 3.8 | 9.0 | 4.0 |

![](http://samlo.t0.ru/igem/stock/image026.png)![](http://samlo.t0.ru/igem/stock/image028.png)

**2.****Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the wells

Ø   95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

**3.****Measured the MQAE fluorescence by microplate reader.**

**4.****Following tubes are inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

**26 Aug (Fri)**

**1.****Measured the OD600 of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.878 | 0.326 | 0.447 | 0.262 | 0.625 |
| Volume to bespinned down (ml) | 2.7 | 7.2 | 5.3 | 9.0 | 3.8 |

**2.****Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the wells. 95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

**3.****Measured the MQAE fluorescence by microplate reader.**

**4.****Following tubes are inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

**28 Aug (Sun)**

**1.****restriction cut of T7, HR (provided by Frankie) and plasmid 1C3**

Ø   HR+T7:
- HR cut by Xbal1 & Pst1
- T7 cut by EcoR1 & Spe1

Ø   HR+1C3, T7+1C3: HR, T7, 1C3 cut by EcoR1 & Pst1

**2.****ligation of HR & T7, HR & 1C3 backbone, T7 and 1C3 backbone respectively**

Ø   10X T4 ligase buffer (2ul) , T4 ligase (1ul) for all tubes

Ø   HR+T7: HR (8.5ul), T7(8.5ul)
- 2 sets, 1 set at 16oC overnight, another set at room temperature, 1hour

Ø   HR+1C3
- 2 sets, 1 set at 16oC overnight, another set at room temperature, 1hour

Ø   T7+1C3
- 1set, at 16oC overnight

Ø   Aliquot 3 tubes with 30ul 10X T4 ligation buffer

Ø   Use 10X T4 ligation buffer in 1.5ml microcentrifuge tubes**

**Week 13****Monday 29 Aug – Sunday 4 Sept**

**29Aug (Mon)**

**1.****PCR purification of ligation product**

**2.****Run gel**

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Lane | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Sample | 1bb ladder | 100bp ladder | HR T7  @16oC | HR T7 @22oC | T7 1C3 @16oC | HR 1C3 @22oC | HR 1C3 @16oC |

**3.****Nanodrop**

**4.****Transformation**

Ø   Use DH5a and BL21 as competent cells for each set

Ø   T71C3 @16oC

Ø   HR1Ce@16oC

Ø   HR1C3@22oC

Ø   6 spread plate stored at incubator

**5.****Following tubes are inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

**30Aug (Tue)**

**1.****PCR purification of HR1C3(16oC****), T71C3(16oC****), T7HR(16oC****)**

**2.****Restriction cut wth**

Ø   T7-HR: Spe1

Ø   T7-HR: EcoR1 & Pst1

Ø   24C double terminator: Xbal1 & Pst1

Ø   pSB1C3: EcoR1 & Pst1

**3.****PCR amplify and purify: HR, T7, HR-T7**

**4.****Run gel to check size**

Ø   Result:
-HR, T7 à correct bands
-HR-T7 à no band

![](http://samlo.t0.ru/igem/stock/image029.jpg)

**5.****Nanodrop****of step 4 product**

|  |  |  |
| --- | --- | --- |
| Sample | ng/ul | 260/280 |
| HR | 12.4 | 1.70 |
| T7 | 7.9 | 1.99 |
| HR-T7 | 8.0 | 1.57 |

**6.****Ligation of step 2 restriction cut product**

Ø   (T7-HR) + (24C double terminator)

Ø   (T7-HR) + (pSB1C3)

**7.****Spread plate**

Ø   HR1C3 DH5a 16oC

Ø   HR1C3 BL21 22oC

Ø   T71C3 DH5a 16oC

Ø   T71C3 BL21 16oC

Ø   Chloride sensor

**8.****Measured the OD600 of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.946 | 0.264 | 0.302 | 0.359 | 0.631 |
| Volume to bespinned down (ml) | 2.5 | 9.0 | 7.9 | 6.6 | 3.8 |

**9.****Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the wells

Ø   95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

**10.****Measured the MQAE fluorescence by microplate reader.**

**11.****Following tubes are inoculated with DH5α pET27b**

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

**31Aug (Wed)**

**1.****PCR purification of ligation product**

Ø   (T7-HR)

Ø   (T7-HR) + (24C double terminator)

Ø   (T7-HR) + (pSB1C3)

**2.****Run gel**

Ø   (T7-HR) , (T7-HR) + (24C double terminator)àno band

Ø   (T7-HR) + (pSB1C3)àwrong size

**3.****Restriction cut and ligation**

Ø   (T7-HR), HR1C3, T71C3

**4.****Run gel with ligation sample**

Ø   HR1C3àcorrect size

Ø   T7-HRàno band

**5.****Gel recovery with HR1C3**

**6.****Pick colony from plate prepared last night**

Ø   T71C3: DH5a

Ø   HR1C3: DH5a & BL21

**7.****PCR amplification of HR & T7**

**8.****Measured the OD600 of the bacteria**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Set up | LB + IPTG without light | NaCl + IPTG with light | NaCl + IPTG without light | KCl + IPTG with light | KCl + IPTG without light |
| OD600(2X) | 0.301 | 0.588 | 0.454 | 0.269 | 0.468 |
| Volume to bespinned down (ml) | 8 | 4.1 | 5.3 | 9 | 5.2 |

**9.****Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each**

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the wells

Ø   95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

**10.****Measured the MQAE fluorescence by microplate reader.**

**1 Sept (Thu)**

**1.****Nanodrop**

**a.****PCR amplified HR & T7**

|  |  |  |
| --- | --- | --- |
| **Sample** | **ng****/ul** | **260/280** |
| HR | 5.4 | 1.83 |
| T7 | 27.1 | 1.73 |

**b.****Ligation**

|  |  |  |
| --- | --- | --- |
| **Sample** | **ng****/ul** | **260/280** |
| HR1C3 @16oC (1) | 16.8 | 1.76 |
| (2) | 18.6 | 1.76 |
| (3) | 12.8 | 1.79 |
| (4) | 16.1 | 1.78 |
| HR1C3 @22oC (1) | 5.7 | 1.96 |
| (2) | 6.9 | 1.69 |
| (3) | 5.2 | 1.77 |
| (4) | 7.5 | 1.64 |

**c.****Gel recovery**

Ø   HR1C3 gel clean: 2.0ng/ul

**2.****Run gel to check size**

Ø  PCR amplified HR & T7 àboth correct

Ø  HR1C3, T71C3àwrong size

Ø  HR-T7àno band

**3.****Restriction cut: HR, T7, pSB1K3**

Ø   HR, T7, 1C3: EcoR1 & Pst1

Ø   HR: Xbal1

Ø   T7: Spe1

**4.****Ligation**

Ø   HR1K3

Ø   T71K3

Ø   HR-T7

Ø   16oC, overnight

**5.****Transformation of gel clean HR1C3 to DH5a**

Ø   Spread 3 plates

**2 Sept (Fri)**

**1.****Transfer lab**

**2.****Pick colony from plate prepared on 1/9**

**3.****Prepared and autoclaved the following solutions**

Ø   500ml LB solution

Ø   250ml LB solution with 1M NaCl

Ø   250ml LB solution with 1M KCl

**3 Sept (Sat)**

**1.****Miniprep****of DH5a with HR1C3**

**2.****Run gel with miniprep product**

**Week 14****Monday 5 Sept – Sunday 11 Sept**

**5 Sept (Mon)**

**1.****Transformation of chloride sensor and 24C terminator**

**2.****Nanodrop****of HR1C3 prepared on 3/9**

|  |  |  |
| --- | --- | --- |
| **sample** | **ng****/ul** | **260/280** |
| 1 | 46.6 | 1.85 |
| 2 | 60.5 | 1.81 |
| 3 | 58.9 | 1.88 |

**6 Sept (Tue)**

**1. Restriction cut: HR1K3, T71K3, T7HR1K3, pSB1C3**

Ø  HR1K3, T71K3, T7HR1K3, pSB1C3: EcoR1 & Pst1

**2. PCR Purification**

**3. Ligation**

Ø  **HR+pSB1C3**

Ø  **T7+pSB1C3**

Ø  **T7-HR+pSB1C3**

**4. Pick colonies of 24C terminator**

**7 Sept (Wed)**

**1. Transformation of HR1C3, T71C3 & T7HR1C3 into DH5alpha**

**2. Miniprep 24C terminator (low concentration)**

**8 Sept (Thu)**

**1. Pick Colonies of HR1C3 & T7HR1C3, T71C3 have no colonies**

**2. Restriction cut T71K3 and pSB1C3**

**3. Ligation T7+1C3**

**4. Transformation of 24C terminator**

**9 Sept (Fri)**

**1. Miniprep HR1C3 and T7HR1C3**

**2. Transformation of T71C3 into DH5alpha**

**3. Pick colonies of 24C terminator**

**10 Sept (Sat)**

**1. T71C3 no colonies**

**2. Miniprep 24C terminator**

**12 Sept (Mon)**

**1. Restriction cut T71K3 and pSB1C3**

**2. PCR Purification**

**3. Ligation T7+1C3**

**13 Sept (Tue)**

**1. Transformation of T71C3 into DH5alpha**

**2. Transformation of HR1C3 and T7HR1C3 into DH5alpha for amplification**

**14 Sept (Wed)**

**1. Restriction cut 24C1AK3 and T7HR1C3**

Ø  **T7HR1C3: EcoR1 & Spe1**

Ø  **24C1AK3: Xba1 & Pst1**

**2. PCR Purification**

**3. Ligation T7HR + 24C + 1C3**

**4. Pick colonies of HR1C3 and T7HR1C3**

**15 Sept (Thu)**

**1. Miniprep HR1C3 and T7HR1C3**

**2. Transformation of T7HR24C1C3**

**3. Characterize the cell adhersion by micromagnetic particles and movement undergone magnetic field attraction as a proof-of-concept model in tubes**

**16 Sept (Fri)**

**1. T7HR24C1C3 no colonies**

**2. Restriction cut T7HR1C3, 24C1AK3 & pSB1T3**

Ø  **T7HR1C3: EcoR1 and Spe1**

Ø  **24C1AK3: Xba1 and Pst1**

Ø  **pSB1T3: EcoR1 and Pst1**

**3. PCR purification**

**4. Ligation T7HR+24C+1T3**

**17 Sept (Sat)**

**1. Transformation of T7HR24C1T3**

**18 Sept (Sun)**

**1. T7HR24C1T3 no colonies**

**2. PCR amplification of T7HR & 24C double terminator using a pair of primers respectively**

**3. PCR purification**

**4. Restriction cut T7HR, 24C and pSB1C3**

Ø  **T7HR: EcoR1 and Spe1**

Ø  **24C: Xba1 and Pst1**

Ø  **pSB1C3: Ecor1 and Pst1**

**5. Run gel and gel recovery**

**6. Ligation T7HR + 24C + pSB1C3**

**19 Sept (Mon)**

**1. Transformation of T7HR24C1C3**

**2. Restriction cut of T7HR1C3 and 24C1AK3**

Ø  **T7HR1C3: Ecor1 and Spe1**

Ø  **24C1AK3: Xba1 and Pst1**

**3. PCR Purified T7HR1C3 cut**

**4. Run gel and gel recovery of 24C cut**

**5. Ligation T7HR24C1C3**

**20 Sept (Tue)**

**1. Pick colonies of T7HR24C1C3 from PCR amplification**

**2. Transformation of T7HR24C1C3 from standard assembly**

**3. Characterize the cell adhersion by micromagnetic particles and movement undergone magnetic field attraction as a proof-of-concept model in tubes (repeat of 15 Sept)**

**21 Sept (Wed)**

**1. Miniprep T7HR24C1C3 from PCR amplification (low concentration)**

**2. Pick colonies of T7HR24C1C3 from standard assembly**

**22 Sept (Thu)**

**1. Miniprep T7HR24C1C3 from standard assembly**

**2, Run gel check size of T7HR24C1C3**

**3. Do DNA sequencing on the biobricks we want to submit**

**Lane 1: 1kb ladder**

**Lane 2: HR1C3 cut by EcorR1 and Pst1**

**Lane3&4: T7HR24C1C3 cut by Ecor1 and Pst1**

**Lane6&7: T7HR1C3 cut by Ecor1 and Pst1**

**Results: correct size of T7HR24C1C3 biobricks Successfully inserted the 24C double terminator.**

**![](http://samlo.t0.ru/igem/stock/image030.jpg)**

**23 Sept (Fri)**

**1. Characterize if biobricks performance same as theoretical HR gene performance on a) Cl- pumping  b) light absoption efficiency  c) regulated protein expression level**

**2. Characterize the cell movement undergone magnetic field attraction in a small-scale proof-of-concept design (repeat of 15 Sept, except same scale as power generation system is being used to demonstrate its movement rate)**

**3. Receive the DNA sequence file of biobricks BBa\_K559000, BBa\_K559001, the sequencing results are correct**

**Series of data has been put in part registry and wiki on our biobricks BBa\_K559000 and BBa\_K559010.**

**24 Sept (Sat)**

**1. Characterize the cell movement undergone magnetic field attraction to be filmed undergone UV light and microscopy**

**Video and photos of the cell movement have been filmed**

**25 Sept (Sun)**

**1. Repeat characterization of the biobricks performance of 23 Sept**

**2. Characterize the cell movement undergone magnetic field**

**Result: All the data are consistent with the work on 23 Sept**

**26 Sept (Mon)**

**1. Repeat characterization of the biobricks performance of 25 Sept**

**2. Receive the DNA sequence file of biobricks BBa\_K559010 , the sequencing result is correct**

**3. Do size check of biobricks BBa\_K559010 again to confirm its size correctness**

**Result: All the data are consistent with the work on 25 Sept, we now have 3 sets of independent results to make error bars for characterization**

**Lane 1: 1kb ladder**

**Lane 3: T7HR24C1C3HR1C3 cut by EcoR1 and Pst1**

**Lane 5: HR1C3 cut by EcoR1 and Pst1**

**![](http://samlo.t0.ru/igem/stock/image032.jpg)**

**We now have correct biobricks to be sent to iGEM part registry!!!**

**27 Sept (Tue)**

**1. Pack all the biobrick samples into iGEM submission format**

**2. Prepare additional sample by doing bacterial transformation to get more DNA of our correct biobricks in case we need it**

**28 Sept (Wed)**

**1. Do series of inoculation, miniprep to get more of our biobricks BBa\_K559001, BBa\_K559001, BBa\_K559010 and nanodrop to measure our biobricks to ensure all the biobricks have enoughamount to store in our lab for future use**

??

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