Team:HZAU-China/WetLab/Labnote

Mixed-Reality CellBidirectional coupling between real and virtual bio-oscillators



Labnote


Dateline

We had seven gene circuits to construct.(as shown in the Figure 1) .After finishing these circuits,we integrated some of them into one plasmid to get two oscillators and one light control system.(as shown in the Figure 2) What’s more,we did three tests to ensure that our system of the real part could work as we expected .


circuit 1: the first circuit of the dual feedback oscillator



circuit 2: the second circuit of the dual feedback oscillator



circuit 3: the first circuit of the quorum sensing oscillator



circuit 4: the second circuit of the quorum sensing oscillator



circuit 5: the third circuit of the quorum sensing oscillator



circuit 6: the first circuit of the light control system



circuit 7: the second circuit of the light control system



plasmid 1: the entire circuit of the dual feedback oscillator



plasmid 2: the entire circuit of the quorum sensing oscillator



plasmid 3: The entire circuit of the light control system




April

Key work: decided our project ; divide into groups; prepare the experimental material

After a month of brainstorming,discussion and three projects presentation,most of the experts and students agree that the project---the mix-reality cell should be our final project. Given the specificity of our project, we divided all team members into three groups, the wetlab group, modeling group and interface group.

At that time,the general framework have been built up.The wetlab group could start to prepare the reagents and materials that required for experiment.

For example:for the basic molecular cloning experiment Solution:LB,antibiotics,LB agar,plates,TAE,glycerol,enthanol,HCl,MgCl2,CaCl2 and so on. Reagents:retriction enzymes(EcoRI XbaI SpeI PstI),DNA polymerase,dNTP,T4ligase,iGEM kits…

Glassware: glass vessel,plastic plate,bottles, measuring cylinder,flasks…


May

Key work: preliminary experiment; formal experiment

We did some preliminary experiments for experimental warming-up. It also aimed to teach some experimental beginners some at-the-bench experimental skills,such as biobrick assembly,Standardize of parts.

Protocol:

1. Two standardize:

-Standardize the mRFP from BBa_J23100’s plasmid backbone through PCR method.

-Standardize the RBS-mRFP-T from BBa_J23100’s plamid backbone through PCR method.

2.Biobrick assembly:

RBS(BBa_B0032)+mRFP

Plac+ RBS+mRFP+T(B0015) (Two standardized parts are provided)



After several weeks’ practices, Almost all wetlab members are familiar with the approach of biobrick assembly and Standardization.

After the preliminary experiment, we went into the formal experiments stage. First of all,we organized all parts related to our project,then get them from the plate kits.


Part

BBa-B0034;BBa-BOO15;BBa-J23100;BBa-J23110;BBa-J23112;BBa-J23114;BBa-J23117;BBa-J23119;BBa-COO12;BBa-c0261;BBa-R0062;BBa-R0063;BBa-I0462;BBa-I14033;BBa-I14034;BBa-I14018 and so on.


June

Key work: circuit 1; circuit 3

Because the two oscillators were mutual independent, we constructed them synchronously. the dual feedback oscillator included two gene circuits(circuit 1,2) and the quorum sensing included three gene circuits(circuit 3-5).

as shown in the figure 1, circuit 1, firstly,we constructed plac/ara-1-RBS-arac-TT and plac/ara-1-RBS-GFP-TT concurrently. then we used biobrick assembly to complete the first circuit of the dual feedback oscillator.

Meanwhile,the circuit3 was constructed in the same approach as well.


July

Key work: circuit 2. 4.6.7

circuit 2 and circuit 4 are simple, so we only spent 10 days on constructing it.

But when we connected the circuit 6,we met some troubles ,we couldn’t get the plasmid of luxR from the Plate Kits 2015 after repeated three times to ensure there were no problem about the operation. Finally, we found this part from the plate kits 2014 successfully.

The way to medals was never easy,before long we got in trouble again .the part bba-k225001 wasn’t provided in the plate kit, but it was a key part for our light control system. What’s more,we can’t find substitute because it is not available with the plates that we had. At one time we communicated with whu-china, we noticed that they have the similar work with us,and they have the part which was exactly what we needed.so we finished constructing the first circuit of the light control system.

The second circuit of the light control system was ordinary and we completed it quickly.


Key work: the entire circuit of the dual feedback oscillator; the entire circuit of the quorum sensing oscillator ; the first rest

Given the real situation in cells ,we placed the circuit 1 and circuit 7 on the plasmid J61002,which have a medium copy number as well as circuit 3 ,circuit 5and circuit 7 .at the same time,the circuit 2 and circuit 6 were placed on the plasmid psb3c5,a vector have a low copy number,as well as the circuit 4 and circuit 6

Now ,all of the main gene circuits were ready .Next,we ran three tests to ensure that our system could work.


September

Key work: the second and the third rest; change the vector to get the standard parts or devices

In Semptember,Our main work is to finish the two remaining detections and change all the backbones of the plasmids which should be submitted on PSB1C3. Two remaining detections: The first one was the influence of light strength to the light sensor. And the other was the characterization of the hybrid promoter Plac/ara-1.

1. First,we cultured the E.coli with light sensor under different light strength till the OD value is near 0.4. Then we used plate reader to detect the fluorescence intensity,and get the corresponding data. (as shown in the flowing)

2. For the promoter test,we applied two kind of E.coli:one of them was ableto Express LacI endogenously,while another didn’t have such ability.Then we cotransformed the constructed circuits for detection:Pcon-RBS-AraC-TT(Pcon is a series of constitutive promoter) and the negative feedback circuit into two kinds of bacteria respectively.We completed the promoter test successfully based on this method.(as show in the characterization)



Daily life

That is when we started to recruit new members, mr.Han was giving a speech on rewirable curcuits. That’s really a good project, almost as good as what we have done this year!

This is the first bonding of our team, come on, “V” is really out of date!

The first Q&A for our project, we got a pretty good photographer, Keji Yan looked much more handsome in this picture than he really is.

iGEM is just for fun, we met some very good friends here our friendship will last even for a life time.
But we should really work hard on it ,after all no pain no gain.




© 2015 Huazhong Agricultural University iGEM Team. All rights reserved.

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