Parts

Our devotion to the community, in reality.

Our New Standard

We made a new standard and we built three parts based on that this year. (BBa_K1431201,BBa_K1431301,BBa_K1431302)

The BbsI restriction enzyme recognize the sequence GAAGAC and cut downstream two bases to six bases. If we make two reverse directions based on BbsI site, we can cut a part and insert another one with seamless. (Mechanism shown below)

Fig.1 The Machanism of Our Standard

Let us make the above sequence as an example. If you want to insert a coding sequence between promoter and polyA, the prefix of forward primer you design must contain GAAGACNNtaaaNNNN and reverse must contain GAAGACNNagttNNNN. The middle two Ns would be any bases for them will be cut off and the four Ns should be begins or ends of the coding sequence. You can get parts by above primers and digest by BbsI restriction enzyme. Then you can insert the parts into promoter and polyA seamless. The PCR product must be such sequence, including the protect bases, shows below.

Fig.2 PCR Product Sequence

The purpose to make such design because we do not know how many or what kind of bases interval between different promoters and polyAs will make the highest transcription efficiency. Just like the data of Kozak consensus sequence, we can measure it by changing the base numbers or kinds and finally find it.

Favorite Parts

Design

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TRE-3G promoter + SV40 PolyA(BBa_K1431301) would be the best parts what we designed this year and also be our favorite parts. The combination of TRE-3G promoter and SV40 PolyA make people insert coding sequence directly without subsection or 3A assembly. The application of our new standard, BbsI site, make a seamless gether which coule test the highest efficiency.

TRE-3G promoter(P_TRE3G) will be switched on after combine with the compound of Tet-On 3G(BBa_K1431101) protein and doxycycline(Dox) mix.

Figure 1.The Tet-On 3G systems allow inducible gene expression only in the presence of doxycycline. When Dox binds, the transactivator undergoes a conformational change allowing it to bind tet operator (tetO) repeats within the
TREG Promoter. The transactivator activates expression through transcription activation domain repeats.

Data

The plasmid transfected into Hela Cell shows below.

pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T

Transfect Hela Cell with different doxycycline(Dox) concentration by lipofectamine3000 and get the data after 30hours.

fluorescence microscope


0 ng/mL doxycycline after 30h	0 ng/mL doxycycline after 30h

100 ng/mL doxycycline after 30h	100 ng/mL doxycycline after 30h

Flow Cytometry


0 ng/mL doxycycline	0.1 ng/mL doxycycline	1.0_ng/mL_doxycycline

10_ng/mL_doxycycline	100_ng/mL_doxycycline	1000_ng/mL_doxycycline

We could find that without Dox, TRE-3G promoter hardly be activated because the fluorescent protein do not expressed. The higher concentration of Dox,the higher number of fluorescent protein expressed. But if the concentration reached 1000 ng/ml, the expression become low, that may be toxic to Hela cell for too high concentration.